Sixty soybean cultivars from Japan and the USA formed five maturity groups (IIb‐Vc) based on number of days from sowing to flowering and number of days from flowering to maturity. Highly significant intervarietal differences in fatty acid composition were found in all the maturity groups, especially in IIc. Stearic and oleic acids showed a larger variation than palmitic, linoleic and linolenic acids. Principal component analysis suggested that the total variation of fatty acid composition depended mainly on the desaturation levels from oleic to linoleic acid. Three cultivars exhibiting unique fatty acid composition, together with a standard cultivar, were examined for the contents of the five fatty acids, as well as crude oil at eight seed‐filling stages. For all four cultivars, it was found that crude oil content increased sigmoidally with advancing filling stage, and that the accumulation patterns of palmitic, linoleic and linolenic acids were similar to that of crude oil. However, the accumulation pattern of stearic acid was different from that of crude oil and divided the cultivars into two distinct groups. For oleic acid, only the cultivar ‘Aburamame’ showed a rapid increase in proportion with advancing filling stage, although not differing markedly in accumulated content from the other cultivars. These results indicate that analysing the accumulation patterns of fatty acids could explain the latent genetic variation in fatty acid composition of soybean seeds.