Seed oil content is a key seed quality trait determining the economic value of rapeseed (Brassica napus L.). However, it is a complex quantitative trait controlled by multiple genes. To this point, its genetic mechanism in rapeseed remains to be revealed. In the present study, we separately identified the quantitative trait loci (QTL) controlling seed oil content of B. napus using three generations of recombinant inbred line (RIL) populations (F_4:5, F_5:6, and F_6:7) derived from a cross of two contrasting parents (M201, a high-oil parent, and M202, a low-oil parent) in four trials. The results indicated that the additive effects may be the primary factors contributing to the variation in seed oil content in B. napus. A total of 15 QTL for seed oil content were mapped. Two of them, namely qOC-A9-3 and qOC-A10, were consistently detected across two and all four environments, respectively. Meanwhile, qOC-A10 showed a large effect on phenotypic variation in seed oil content. The stability and significance of qOC-A10 was also validated in the near isogenic lines (NILs-qOC-A10) developed from the RIL population (F_4:5) using marker-assisted selection. The qOC-A10 is of particular interest for further fine mapping and map-based cloning.