Global food security and sustainability can be enhanced with increased production and use of hemp seed ( Cannabis sativa L.) that not only provides healthy sources of protein and oil, but the whole plant can be used for fiber, especially new applications including high-performance textiles. There is limited knowledge of the variation in hemp seed for health and consumer traits, such as the complex plant polysaccharides comprising dietary fiber. To fill this gap, seed from 20 hemp cultivars and advanced breeding lines were analyzed for a variety of traits including complex carbohydrates, protein, lipids, heart percent, phytate and lignin. The analyses revealed different polysaccharides in hull and heart fractions, with hulls varying in cellulose (22.0–36.7%) and xylan (5.7–17.1%) whereas heart fractions contained xyloglucan and pectin, and comprised different proportions of heart and hull. Staining revealed that cellulose and lignin are localized in hull not heart tissue. Protein content of hemp seed ranged from 19.5 to 26.9% and Coomassie staining showed that cells of hemp hearts contain numerous protein bodies but few starch granules. Analysis of selected lines confirmed that hemp hearts are low in starch (<2%). Lipid content varied from 26.6 to 37.8%, with the omega-6:3 ratio varying from 2.1 to 4.9 with seven lines having a ‘desirable’ omega-6:3 ratio of less than or equal to three. This research provides breeders with additional traits to consider for maximizing consumer satisfaction and human health. • Hemp cultivars and breeding lines have variation for omega-3 α-linolenic acid. • Compositional analysis of heart (kernel) and hull (husk) fractions of hemp seed. • Hemp seed contain non-cellulosic dietary fiber including xylan, xyloglucan & pectin. • Hemp hearts are low in starch (<2%) and soluble sugars. • Hemp proteins are found in protein bodies.