Oil content is a primary trait in soybean and determines the quality of soy food, feed and oil product. Increasing oil content is a major objective of soybean breeding. The aims of the present study were to identify quantitative trait loci (QTLs) and epistatic QTLs associated with oil content in soybean seed by using 129 recombinant inbred lines derived from a cross between cultivar Dongnong 46 (oil content 22.53%) and the semi-wild line L-100 (oil content 17.33%). Phenotypic data were collected from 10 tested environments including Harbin in the years 2012–15, Hulan in 2013–15 and Acheng in 2013–15. A genetic linkage map including 213 simple sequence repeat markers in 18 chromosomes (or linkage groups) was constructed, covering ∼3623.39 cM. Seven QTLs, located on five chromosomes (or linkage groups), were identified to be associated with oil content, explaining 2.24–17.54% of the phenotypic variation in multi-environments. Among these identified QTLs, five (qOIL-2, qOIL-4, qOIL-5, qOIL-6 and qOIL-7) were detected in more than five environments. Seven QTLs had additive and/or additive × environment interaction effects. QTLs with higher additive effects were more stable in multi-environments than those with lower additive effects. Moreover, five epistatic, pairwise QTLs were identified in different environments. The findings with respect to genetic architecture for oil content could be valuable for marker-assisted selection in soybean breeding programs for high oil content.