Epistasis is considered to be an important feature of quantitative performance in self-pollinated (homozygous) crop species. A bi-parental segregating population between two early maturity soybean parents (Ma.Belle x Proto) consisting of 82 F2-families had initially been analysed with a large number of polymorphic SSR markers, and QTL effects in seed characters had been reported. In the F6 generation, 5 to 8 different lines were derived from each F2 family, and a total of 530 homozygous lines was evaluated phenotypically in four environments and re-genotyped using 29 SSR markers associated with QTLs in the initial analysis. Using analysis of variance for testing of associations, about 10 QTLs were found for 1000-seed weight and several others for seed protein and oil content, respectively. The presence of 1000-seed weight QTLs on soybean linkage group O was confirmed. Two-way analysis of variance revealed a number of highly significant interactions between markers, indicating epistatic gene interaction. Significant interactions have been found both between QTL regions as well as between regions without a significant main effect. Similar to QTL main effects, some epistatic QTL effects were stable across environments, whereas for others a significant epistasis x environment interaction effect was present. In 1000-seed weight, the phenotypic effect of particular epistatic QTLs was clearly larger than the effect of the main QTLs found. The present results suggest, that epistatic gene interaction has a considerable impact on the genetics of 1000-seed weight of soybean.