This study comprehensively characterized the chemical composition of 29 different varieties of industrial hempseed (Cannabis sativa L.). The analysis included fatty acid composition, amino acid profile, mineral content, and cannabinoid content. All hempseed varieties were milled to obtain whole hempseed flour, and significant differences (p < 0.05) were observed in all measured parameters. The proximate composition showed that crude protein and fat contents ranged from 21.6–28.9% and 21.1–35.7%, respectively. Fatty acid profiles revealed linoleic acid as the major component (52.79–57.13%), followed by α-linolenic acid (12.62–20.24%) and oleic acid (11.08–17.81%). Essential amino acids were detected in all varieties, with arginine being the most abundant (12.66–17.56 mg/100 g protein). Mineral content showed substantial variations, with potassium (509.96–1182.65 mg/100 g) and iron (5.06–32.37 mg/100 g) being the main macro- and microminerals, respectively. Cannabinoid content was low, with tetrahydrocannabinidol (THC) detected in only five varieties. The nutritional composition of hempseeds, including hulls, makes them suitable for human and animal diets as a beneficial novel ingredient. The study highlights the potential of hempseeds as a valuable source of nutrients and bioactive compounds, emphasizing the importance of further research to quantify antinutritional compounds, phenolic compounds, and bioactive peptides.This study comprehensively characterized the chemical composition of 29 different varieties of industrial hempseed (Cannabis sativa L.). The analysis included fatty acid composition, amino acid profile, mineral content, and cannabinoid content. All hempseed varieties were milled to obtain whole hempseed flour, and significant differences (p < 0.05) were observed in all measured parameters. The proximate composition showed that crude protein and fat contents ranged from 21.6–28.9% and 21.1–35.7%, respectively. Fatty acid profiles revealed linoleic acid as the major component (52.79–57.13%), followed by α-linolenic acid (12.62–20.24%) and oleic acid (11.08–17.81%). Essential amino acids were detected in all varieties, with arginine being the most abundant (12.66–17.56 mg/100 g protein). Mineral content showed substantial variations, with potassium (509.96–1182.65 mg/100 g) and iron (5.06–32.37 mg/100 g) being the main macro- and microminerals, respectively. Cannabinoid content was low, with tetrahydrocannabinidol (THC) detected in only five varieties. The nutritional composition of hempseeds, including hulls, makes them suitable for human and animal diets as a beneficial novel ingredient. The study highlights the potential of hempseeds as a valuable source of nutrients and bioactive compounds, emphasizing the importance of further research to quantify antinutritional compounds, phenolic compounds, and bioactive peptides.