The accumulation of seed mass in soybean is affected by both genotype and environment. The aim of the present study was to measure additive, epistatic and quantitative trait locus (QTL)×environment (QE) interaction effects of QTLs on the development of 100-seed weight in a population of 143 F5 derived recombinant inbred lines (RILs) developed from the cross between the soybean cultivars ‘Charleston’ and ‘Dong Nong 594’. Broad-sense heritability of 100-seed weight from 30 days (30D) to 80D stages was 0·58, 0·52, 0·62, 0·60, 0·66 and 0·57, respectively. A total of 17 QTLs with conditional additive (a) effect and/or conditional additive×environment interaction (ae) effect at specific stages were identified in ten linkage groups by conditional mapping. Of them, only 4 QTLs had significant a effect or ae effect at different stages of seed development. Among QTLs with significant a effect, five acted positively and six acted negatively on seed development. A total of 35 epistatic pairwise QTLs of 100-seed weight were identified by conditional mapping at different developmental stages. Five pairs of QTL showed the additive×additive epistatic (aa) effect and 16 QTLs showed the aa×environment interaction (aae) effect at the different developmental stages. QTLs with aa effect as well with their environmental interaction effect appeared to vary at different developmental stages. Overall, the results indicated that 100-seed weight in soybean is under developmental, genetic and environmental control.