The industrial hemp study was established at three locations in Missouri: George Washington Carver (GWC) farm in Jefferson City (Longitude: 38.5322° N; Latitude: - 92.1333° W), Sikeston Agri-Park in Sikeston (Longitude: 36.8831° N; Latitude:-89.5878° W) and Washington, Missouri (Longitude: 38.5581° N; Latitude: -91.0121° W) (Supplementary 1, Table 1). The experimental sites represented distinct agro-ecological conditions (Table 2, Figure 1). At Carver Farm, soils were classified as Menfro silt loam, well-drained, with a pH range of 6.0-6.5, and characterized by deep, loess-derived profiles with moderate organic matter. Sikeston soil was Alfisols (silty loam), well-drained, with pH 6.1-6.5, typical of the Missouri Bootheel row-crop region. Washington soils were loess-derived silty loam, moderately well-drained, slightly more acidic (pH 6.0-6.2), and exhibited variable moisture retention (Table 1). Thirty-two industrial hemp cultivars, consisting of both fiber and dual-purpose types, sourced from domestic and international seed suppliers were studied (Table 2). In the 2021 summer growing season, fifteen cultivars were selected and evaluated at two sites: GWC farm (Jefferson City) and Sikeston. In 2022, the study expanded to include twenty-two cultivars grown across all three locations. Based on performance data from the previous two years, the top eight cultivars were selected for further evaluation during the 2023 summer growing season.

Industrial hemp cultivars were evaluated in a randomized complete block design (RCBD) with two replications per location (Tamang et al., 2025; Babaei and Ajdanian, 2020; Papastylianou et al., 2018). Prior to sowing, all field sites underwent standard land preparation, consisting of two passes of conventional tillage followed by culti-packing to establish a uniform and firm seedbed. Basal fertilization was applied at a rate of 70-60–40 kg ha⁻¹ of nitrogen (N), phosphorus (P), and potassium (K), respectively, in accordance with regional agronomic recommendations for hemp cultivation (Kaur et al., 2023; University of Missouri Extension, ). Irrigation management was designed to simulate farmer field conditions, where cultivation is predominantly rainfed. Supplemental irrigation was only applied at critical growth stages (early vegetative, flowering, and seed set) when rainfall was insufficient or drought stress indicators were observed (leaf wilting, soil moisture depletion). Irrigation decisions were based on visual field assessments, with water requirement estimates informed by FAO modeling approaches (Food and Agriculture Organization (FAO), 1992) and supported by region-specific studies, such as Thevs and Aliev (2022), which reported ~350 mm seasonal water consumption for hemp under temperate conditions. Sowing was performed using a John Deere 1590 No-Till Drill, calibrated at a seed drop setting of 22 to accommodate the relatively larger seed size typical of industrial hemp. The seeding rate was approximately 56 kg ha^-1 of viable seed, consistent with industry recommendations for fiber- and dual-purpose hemp production (45–65 kg ha⁻¹) (Cherney and Small, 2016; Giannoulis et al., 2024). However, following seed viability assessments (germination tests) conducted prior to sowing each year, cultivar-specific seeding rates were adjusted to compensate for differences in germination percentage (GP) (Table 3). Seeding rate was then calculated using the formula (International Seed Testing Association (ISTA), 2022):

Where:

Planting dates, plot dimensions, row spacing, and planting depth for each site and year are summarized in Table 4 to give an overview of the field setup used for industrial hemp trials conducted across three Missouri locations between 2021 and 2023.

In 2023, severe flooding events at the Sikeston and Washington locations affected planting activities, resulting in incomplete datasets from these sites. Accordingly, data from affected plots were excluded from subsequent statistical analyses.

Phenotypic traits were recorded throughout the growth cycle and included emergence rate, flowering time, plant density, plant height, stem diameter, biomass, and yield. Plant density was determined by counting all plants within the two 1-m² area within the plot (totaling 2 m² per plot). For plant height, twelve plants were randomly selected from each plot. Stem diameter was measured on the same twelve plants, with two measurements per plant: one taken 12.7 cm above the soil surface (basal diameter), and the other 12.7 cm below the apical tip (upper diameter). Germination performance (GP) was evaluated under controlled greenhouse and laboratory conditions, without any pre-treatment of seeds (e.g., scarification or chemical priming). For each cultivar, 100 seeds were sown in three replications under uniform conditions. Germination percentage (GP) was then calculated using the standard formula (Gadissa et al., 2022; Shah et al., 2021):

Emergence rates were visually scored on a 1–5 scale (1 = 0%, 5 = 100%) between 5 and 14 days after planting. Flowering time was recorded when 50% of the plants within a plot exhibited visible flowers.

Biomass was assessed at full maturity by uprooting entire plants and recording their fresh weight. The samples were then dried for 2–3 weeks at 49 °C to obtain constant dry weight. Once a constant moisture content was achieved, the dry weight was recorded. Biomass percentage was then calculated using the formula:

Climatic data, including temperature, precipitation, and humidity, were collected throughout the growing seasons using on-site weather stations (Campbell Scientific Inc., Logan, UT, USA) (Figure 1).

To evaluate cultivar performance across locations, we selected a core subset of eight industrial hemp cultivars from the total set evaluated. These cultivars were chosen because they consistently produced uniform stands and progressed through phenological stages at all three experimental sites, even under challenging environmental conditions (e.g., flood). Their stable performance across diverse environments made them suitable for valid and reliable multi-location comparative analysis. The analysis for this subset focused on key agronomic parameters, including stem diameter, plant height, plant density, whole-plant biomass, and fiber yield. To account for environmental variation across sites, data were normalized using Z-score standardization, following the method described by Cheadle et al. (2003), using the formula:

Where:

Agronomic and yield data were log-transformed prior to statistical analysis to normalize distributions and stabilize variances. However, original (non-transformed) values were retained for graphical presentations and comparison of cultivar trends. Statistical analyses were conducted using RStudio (version 2022.07.1 Build 554) and Minitab^® version 22.1. The general linear model (GLM) procedure for analysis of variance (ANOVA) was used to test the effects of cultivar, location, year, and interactions. Means were separated using Tukey’s Least Significant Difference (LSD) at a 95% confidence level (α = 0.05). Correlation coefficients were computed according to the method described by Pearson (1895) (Figure 2).

Moreover, a post-hoc power analysis was performed to evaluate the ability of the experimental design to detect meaningful differences among cultivars. The analysis was conducted separately by site and year using residual mean square error (MSE) values obtained from one-way ANOVA models (trait- cultivar).

The minimum detectable difference (MDD) was calculated for plant density and biomass (kg m⁻²) at 80% statistical power and a significance level of α = 0.05 using the following equation:

where t values correspond to the student’s t distribution, df represents residual degrees of freedom, and n is the number of replications per cultivar.

Fresh leaf samples from GWC farm-Jefferson City and Sikeston were collected and immediately flash-frozen in liquid nitrogen and stored at -80 °C until further analysis. The flash frozen samples were freeze-dried and ground into a fine powder. A sample size of 200 mg was used for extraction, with three solvent mixtures tested: 100% methanol, methanol: water (9:1), and methanol: acetonitrile (9:1) (Tzimas et al., 2024). The powdered samples were mixed with 5 mL of solvent, vortexed for 10 minutes, and sonicated for 60 minutes. After centrifugation at 4500 rpm for 10 minutes, the supernatant was filtered through a 0.22 μm PTFE syringe filter before High-Performance Liquid Chromatography (HPLC) analysis.

HPLC analysis was conducted using a Shimadzu Hemp Analyzer, injecting 10 μL of the extract under a column temperature of 35 °C for 10 minutes. Cannabinoid concentrations, including THC and CBD, were determined using calibration standards and peak area ratios (Table 5). A mobile phase system consisting of phosphoric acid in water, acetonitrile, and methanol-based solvents were used for system stabilization. Methanol: acetonitrile (9:1 v/v) mixture was employed based on its proven efficiency in solubilizing phenolic compounds in hemp leaf tissues. This combination of methanol and acetonitrile offers high polarity and low viscosity, which enhances the extractability of both structural and biochemical constituents, including cannabinoids and secondary metabolites for downstream analysis. While this ratio was optimized within the context of the current study, the decision was guided by previous research demonstrating its effectiveness in extracting a broad range of phytochemicals from Cannabis sativa (Tzimas et al., 2024). Statistical analysis was performed using RStudio (version 2022.07.1 Build 554). A one-way ANOVA was conducted to evaluate differences in cannabinoid concentrations among cultivars.

Monthly weather conditions varied notably across the years and locations. Air temperatures ranged from 10 °C to 25 °C, with higher means observed in 2023, particularly at GWC farm and Washington. In 2022, both the Sikeston and Washington sites recorded the highest total rainfall observed during the study period. Relative humidity remained above 70% overall, though Washington experienced a decline in 2022. Soil moisture was highest at GWC farm in 2022–2023 but notably reduced in Washington in 2023. Soil temperatures mirrored air temperature trends, reaching their highest in 2023 across all sites, indicating warmer growing conditions during that season.

Germination percentages among the thirty-two industrial hemp cultivars varied across the three experimental years (Table 3), reflecting both genetic diversity and potential differences in seed quality and viability. In 2021, germination ranged from as low as 1% in Grandi to 78.7% in Ferimon, with a grand mean of 41.55%. Cultivars such as Bialobrzeskie (56.7%), Fibror 79 (68.1%), and Jinma (64.5%) performed well above the grand mean, whereas Anka (6.5%), Katani (7.5%), and CRS-1 (8.6%) showed extremely poor viability (<10%).

In 2022, overall germination improved substantially (grand mean 64.47%), with top-performing cultivars including Bialobrzeskie (92.2%), Jinma (90.9%), and Fibror 79 (89.9%). Poor-performing lines included BVL5 (0%) and Fibranova (24%). The 2023 results, though covering fewer cultivars, showed the highest overall viability (grand mean 73.31%), with Futura 83 (88%), Bialobrzeskie (81.1%), and Jinma (85.6%) maintaining strong performance. Statistical analysis confirmed significant differences among cultivars in each year (p< 0.05), with least significant differences (LSD) of 1.86, 2.18, and 2.19 for 2021, 2022, and 2023, respectively. European cultivars generally exhibited high germination rates, consistent with previous studies (Amaducci et al., 2015; Tang et al., 2022), whereas poor performance in certain cultivars likely resulted from genetic limitations, seed viability or dormancy. The associated seeding rate calculations, adjusted for germination percentage, revealed the practical implications of seed viability on field establishment. For instance, cultivars with high viability such as Ferimon and Felina 32 required ~70 kg ha^-1o achieve the target viable seed rate, while low-performing cultivars such as Anka and Katani required >747 kg ha^-1 rendering them impractical for commercial planting. These findings underscore the importance of pre-sowing germination testing and cultivar selection to ensure optimal stand establishment and resource efficiency in industrial hemp production.

In 2021, fifteen industrial hemp cultivars were evaluated at two agroecological sites, GWC farm (Jefferson City) and Sikeston, Missouri, revealing significant variation (P< 0.001) in all measured traits, including emergence rate, plant height, stem diameter, plant density, and biomass (Tables 6, 7).

Biomass, plant height, and stem diameter exhibited inter-annual and inter-cultivar variability across all three study sites (GWC, Sikeston, and Washington) over the 2021–2023 growing seasons (Figures 4–6).

Environmental conditions strongly influence hemp growth, particularly during early establishment and flowering (Struik et al., 2000; Amaducci et al., 2015). Across the three-year study, variation in temperature, rainfall, humidity, and soil moisture significantly affected cultivar performance. Reduced biomass at Washington in 2023 was likely driven by high evapotranspiration and limited soil moisture, consistent with stress-related constraints on canopy development and carbon assimilation reported by Lisson et al. (2000) and Ortmeier-Clarke et al. (2023) under water-limited conditions. Conversely, favorable conditions at Sikeston in 2022, characterized by higher rainfall, moderate temperatures, and greater humidity, likely enhanced biomass accumulation, particularly for high-yielding cultivars such as Jinma. This agrees with findings by Campiglia et al. (2017) and Adesina et al. (2020), who showed that hemp growth is optimized under moderate temperatures (20–25 °C) and adequate soil moisture. Similar location-by-year effects across U.S. agroecological zones were reported by Williams et al. (2025) and Stack et al. (2021), emphasizing the importance of genotype–environment matching. Accordingly, climate-adapted cultivar selection combined with locally responsive agronomic practices is essential for sustaining hemp productivity under increasing climatic variability (Panday et al., 2025).

Hemp emergence is influenced by genetic and environmental factors such as seed vigor, soil moisture, and temperature (Poudel et al., 2020; Amaducci et al., 2015; Campiglia et al., 2017). Higher emergence at the GWC farm in 2022 was likely due to moderate temperatures and adequate moisture that favored uniform seedling establishment. In contrast, lower emergence at Washington may reflect elevated soil temperatures and moisture deficits that constrain early growth. Cultivars including Jinma, Felina 32, and Ferimon showed stable emergence across environments, indicating greater seedling vigor and tolerance to early-season stress. The strong correlation between emergence and plant density (r = 1.00) aligns with Panday et al. (2025), reinforcing the role of uniform emergence in maximizing biomass production. These results highlight the importance of environment-specific cultivar selection and planting strategies in stress-prone regions.

Flowering time variation among hemp cultivars is governed by photoperiod sensitivity and genotype-environment interactions, both of which significantly impact regional adaptability and harvest scheduling (Zhang et al., 2021; Petit et al., 2020). Late-flowering cultivars such as Jinma, developed at lower latitudes (e.g., China), typically require shorter photoperiods to trigger reproductive development. In contrast, northern genotypes like Ferimon and USO 31 are either photoperiod-neutral or have reduced sensitivity, leading to earlier flowering under Missouri’s extended summer daylight conditions (Van der Werf et al., 1994; Lisson et al., 2000). Our field data confirm this divergence, with Jinma flowering at approximately 105 DAPS: well within the region’s frost-free window. No frost damage was observed at any site during the three-year study period, as air temperatures remained above freezing in October, and soil temperatures consistently exceeded 15 °C during the flowering-to-harvest phase (Figure 1). This climatic stability confirms that late-flowering cultivars like Jinma are viable in central and southern Missouri. However, in more northern regions or in years with early frost onset, such cultivars could be at greater risk of biomass and quality loss, particularly in fiber-production systems. Genetic studies by Toth et al. (2022) and Dowling et al. (2024) underscore the influence of FLOWERING LOCUS T (FT) homologs and other photoperiod-regulatory genes in controlling flowering time across latitudes, supporting the phenological variation we observed. These findings align with previous work by Amaducci et al. (2008), who emphasized the importance of synchronizing cultivar phenology with regional frost-free periods to ensure reliable production and fiber quality.

Significant variation in plant density was observed across cultivars and locations, reflecting the combined influence of emergence rate, seedling vigor, and environmental conditions. Higher plant densities at Sikeston in 2022 were likely supported by favorable rainfall and soil moisture conditions, promoting uniform germination. In contrast, lower densities recorded at Washington were consistent with the site’s drier and warmer conditions. Cultivars such as Futura 83, Felina 32, and Jinma demonstrated consistently high stand densities, indicative of strong early vigor and adaptability. These attributes are particularly beneficial for fiber-production systems, where dense stands promote vertical growth and reduce lateral branching (Small and Marcus, 2002). Conversely, reduced densities in cultivars like USO 31 and Santhica 70 at Washington suggest the need for improved seed quality, adjusted seeding rates, or site-specific agronomic practices. These findings support previous recommendations by Tang et al. (2017), suggesting higher seeding densities (90–150 plants m⁻²) for fiber production and lower densities (30–75 plants m⁻²) for seed-production systems (Tamang et al., 2025), to balance stem quality and minimize intra-specific competition. Overall, the correlation analysis between agronomic and climatic variables demonstrated that hemp growth and yield performance were closely linked to environmental conditions (Figure 2). This reinforces the role of genotype-environment interactions in shaping hemp productivity across diverse field sites and growing seasons.

Jinma, a tall Chinese fiber-type cultivar, consistently exhibited superior aboveground biomass and fiber yield across locations and years, demonstrating strong genotypic stability and broad agroecological adaptability within the southern U.S. Midwest. Its performance was particularly robust at the GWC farm, where fiber yields reached approximately 5.8 kg m⁻² (~ 3–5 t acre⁻¹), and remained comparatively high at Sikeston and Washington despite contrasting environmental conditions. These results are especially notable given the limited availability of region-specific management guidelines for fiber hemp following its recent reintroduction in the United States.

When contextualized against other bast fiber crops, Jinma’s productivity is comparable to reported kenaf yields (5–10 t acre⁻¹) and substantially exceeds typical flax bast fiber yields (~0.8 t acre⁻¹) under U.S. field conditions (Austin et al., 2024; Arefin et al., 2021). Other cultivars, including Fibror 79 and Ferimon, exhibited moderate biomass and fiber potential but showed greater sensitivity to environmental variability, particularly under suboptimal conditions. The reduced biomass and fiber yields observed in 2023, especially at the Washington site, were likely driven by early-season flooding followed by elevated summer temperatures, which constrained stand establishment and vegetative growth. These trends are consistent with previous studies indicating that tall, fiber-oriented cultivars maximize biomass and fiber accumulation under favorable temperature, moisture, and plant density conditions (Amaducci et al., 2015; Tang et al., 2017). Jinma’s high productivity can be attributed to complementary morphological traits, such as tall stature, thick stems, and strong early vigor, that enhance resource-use efficiency and fiber yield. Its stable performance across diverse environments supports its suitability for resilient fiber-based production systems, while reduced performance under stress underscores the importance of site-specific cultivar selection (Finnan and Burke, 2013).

Plant height is a critical determinant of stem biomass, bast fiber and hurd yield in fiber hemp. Among the cultivars evaluated, Jinma and Fibror 79 consistently achieved the tallest height across years and locations, reflecting both their genetic selection for fiber production and adaptability to variable environments. Favorable conditions at GWC and Sikeston in 2022, characterized by moderate temperatures and sufficient soil moisture, further promoted elongation, consistent with findings by Williams et al. (2025), who reported strong genotype × environment effects on hemp height in the Midwestern region, US. A strong positive correlation between plant height and biomass reinforces the role of vertical growth in fiber yield potential, corroborated by previous work (Small and Marcus, 2002; Salentijn et al., 2015; Musio et al., 2018; Rehman et al., 2021; Ortmeier-Clarke et al., 2023). In contrast, Washington’s hotter and drier conditions in 2023 suppressed stem elongation across all genotypes, an outcome attributed to heat and drought stress that limits internodal development (Campiglia et al., 2017; Panday et al., 2025).

Photoperiod-temperature interactions also modulated plant height variation, particularly in photoperiod-sensitive cultivars, which showed strong responses to day length and growing degree accumulation (Petit et al., 2020; Zhang et al., 2021). These dynamics underscore the importance of matching cultivar phenology to local climatic regimes. Although Bialobrzeskie maintained reasonable emergence and stand density (Figure 6), its moderate height reduced its suitability for fiber-production systems. Shorter cultivars tend to exhibit limited internodal elongation and reduced bast fiber proportion, making them more appropriate for dual-purpose or seed-oriented production where earlier flowering and easier mechanical processing are prioritized (Rahemi et al., 2021; Ortmeier-Clarke et al., 2023). Overall, these findings highlight the need for cultivar-environment matching in hemp cropping systems. Tall, late-flowering genotypes like Jinma are best suited to regions with extended growing seasons and sufficient late-season moisture, while compact cultivars may perform better in stress-prone or short-season environments. As noted by Panday et al. (2025), optimizing spacing, moisture availability, and temperature during vegetative stages is essential for maximizing height and fiber biomass.

Stem diameter is a critical morphological trait in fiber hemp, closely linked to structural biomass, bast fiber yield, and mechanical processing efficiency. Across all environments and years, Jinma consistently exhibited the thickest stems, even under suboptimal moisture conditions at the Washington site, suggesting robust genetic vigor and potential water-use efficiency. These results support earlier findings by Tang et al. (2017), Petit et al. (2020), and Musio et al. (2018), which highlight thicker stems as indicators of enhanced bast fiber content and total stem biomass in fiber-bred cultivars. A strong positive correlation was observed between stem diameter and both plant height and biomass production, reinforcing the interconnected nature of architectural traits and their cumulative impact on fiber productivity. This aligns with the understanding that stem thickness contributes significantly to both yield and harvest quality in fiber hemp. Inter-annual variation also influenced diameter patterns. In 2022, reduced variability across cultivars pointed to favorable and uniform moisture and temperature conditions (Panday et al., 2025), which may have buffered environmental stress. Conversely, the 2023 decline in stem diameter across all genotypes likely reflects climate-induced stress, including heat and late-season drought, factors known to suppress cambial activity and secondary thickening (Campiglia et al., 2017; Ortmeier-Clarke et al., 2023). Cultivars such as Bialobrzeskie and USO 31, which consistently produced narrower stems, may be better suited for dual-purpose or seed-oriented systems, where lower lignification can facilitate mechanical harvesting and decortication (Rahemi et al., 2021; Petit et al., 2020). These findings underscore the importance of matching cultivar architecture to production goals and environmental constraints, particularly in variable Midwestern agroecosystems.

Although the multi-location, multi-year experimental design across Missouri strengthened the environmental representativeness of our findings, we acknowledge that the use of only two replications per site may have reduced the overall statistical power of the study and potentially mask minor differences among cultivars. Nonetheless, the consistent trends observed across years and location, particularly for high-performing cultivars such as Jinma and Fibror 79, further support the robustness of our findings. Moreover, power analysis indicates that the experimental design was adequate to detect biologically meaningful differences among cultivars for key traits. At the Washington site, the minimum detectable difference was estimated at 5.34 plants m⁻² for plant density and 0.62 kg m⁻² for biomass, thresholds that are well within the observed range of cultivar variation.

These results suggest that large and agronomically relevant differences, such as those observed between high-performing cultivars (e.g., Jinma, Puma) and low-performing cultivars, were detected reliably. However, smaller differences among intermediate cultivars may have gone undetected, particularly under environmentally stressful conditions such as flooding in 2023. It is important to acknowledge that the 2023 flooding events at the Sikeston and Washington sites resulted in the exclusion of complete datasets from those environments. This data loss reduced the temporal balance of the three-year dataset and may have limited our ability to fully capture inter-annual variability across all locations. However, consistency in cultivar performance observed between 2021 and 2022, as well as the unaffected 2023 GWC site, indicates that the exclusion did not materially alter overall conclusion. However, future experiments should incorporate higher replication numbers (≥3 per site) or spatially augmented designs to enhance precision and reliability of genotype × environment interaction analyses.

The observed variation in THC concentration among cultivars across environments highlights the complex genotype × environment (G×E) interactions that govern cannabinoid expression. Warmer temperatures, delayed harvest timing, and abiotic stressors such as drought have been shown to elevate THC accumulation, likely by extending the flowering phase or activating stress-induced biosynthetic pathways (Chandra et al., 2020; Berthold et al, 2020). Despite these fluctuations, cultivars such as Fibror 79, Futura 83, and Santhica 70 consistently remained below the legal THC threshold, affirming their suitability for compliant commercial production. The stable performance of these cultivars aligns with recent profiling studies, which confirm their low-THC genotypic backgrounds (Lindekamp et al., 2024; Nahler et al., 2019: Süzerer et al., 2023). At the same time, elevated CBD concentrations in cultivars like Bialobrzeskie, Fibror 79, Felina 32, and Ferimon suggest strong dual-purpose potential, offering value for both fiber and phytochemical applications. This confirms the importance of multi-trait selection strategies that integrate biomass performance with stable cannabinoid profiles (Small, 2015; Adesina et al., 2020).

Year-to-year and site-specific variation in CBD concentrations (Figure 8) highlights the strong influence of genotype × environment interactions. Higher CBD accumulation at Sikeston likely reflects favorable soil or microclimatic conditions relative to GWC farm, while elevated CBD levels in cultivars such as Bialobrzeskie and Fibror 79 in 2024 suggest responsiveness to improved agronomic or climatic conditions, consistent with Andre et al. (2016). In contrast, fiber-dominant cultivars including Jinma, Futura 83, and Santhica 70 maintained consistently low CBD levels, confirming their limited phytochemical potential and suitability for compliant fiber production. These inter-cultivar and inter-site patterns have direct implications for value chain specialization: cultivars with low cannabinoid accumulation are best suited for fiber systems, whereas genotypes expressing moderate to high CBD levels (e.g., Bialobrzeskie, Ferimon, Felina 32, and Fibror 79) offer potential for dual-purpose or phytochemical-oriented production. Overall, integrating agronomic performance with cannabinoid stability provides a practical framework for cultivar selection across industrial and pharmaceutical hemp value chains. Collectively, these findings emphasize the need for multi-year, multi-location evaluations of hemp cultivars, especially under variable climatic conditions, to ensure cannabinoid consistency and regulatory compliance. Advances in rapid cannabinoid quantification technologies, such as NIR spectroscopy (Jarén et al., 2022), and molecular characterization of biosynthetic enzymes like THCA and CBDA synthase (Van Bakel et al., 2011), offer promising avenues for precision phenotyping and cultivar certification. Moreover, the concept of sustainability in hemp production can be further interpreted through the input–output balance observed in this study. Industrial hemp demonstrated high biomass productivity under moderate input conditions, specifically, basal fertilization of 70-60–40 kg ha⁻¹ (NPK) and largely rainfed irrigation supplemented only at critical growth stages. Across sites, cultivars such as Jinma and Fibror 79 produced between 6 and 12 t ha⁻¹ of dry fiber yield, translating to an estimated nitrogen-use efficiency of 85–120 kg biomass per kg N applied, which is comparable or superior to conventional fiber crops like cotton and kenaf (Campiglia et al., 2017; Kaur et al., 2023). These results highlight hemp’s capacity to achieve substantial biomass gains with limited nutrient and water inputs, thereby reducing its environmental footprint. Moreover, its short growth cycle and high biomass production contribute to atmospheric carbon sequestration, reinforcing its role as a climate-smart rotation as well as cover crops. Although this study did not include a full life-cycle assessment, the observed yield-to-input ratio provides a practical indication of hemp’s potential as a sustainable, resource-efficient crop for Midwestern production systems.