Seed isoflavone content is influenced by environment and genotype, but seed fractions such as hypocotyls and cotyledons display highly contrasted isoflavone contents and compositions. Moreover, the isoflavone content of hypocotyls appears less influenced by environment than that of cotyledons. The purpose of this study was to better understand isoflavone distribution between the two parts of the embryo during seed maturation. The accumulation kinetics of individual and total isoflavones in hypocotyls and cotyledons were recorded in three growth conditions (two years in greenhouse, and one year in the field) in four soybean [Glycine max (L.) Merr.] cultivars having contrasted isoflavone contents and compositions in their seed fractions, from 20 days after flowering (DAF, R5 stage) to seed maturity (R8 stage). As soon as 20 DAF, early isoflavone accumulation was observed in hypocotyls, whereas the isoflavone accumulation the in cotyledons began at R6 stage, after hypocotyl content had reached a plateau. Depending on the growth conditions, a continuous accumulation up to maturity was observed in cotyledons, resulting in a fourfold increase of the total isoflavone content. But whatever the growth conditions, isoflavone accumulation in hypocotyls was unchanged, suggesting that hypocotyl isoflavone content could mainly be under genotypic control. This study clearly shows that isoflavone synthesis and accumulation result from highly differentiated pathways in hypocotyls and cotyledons.