BackgroundIncreasing seed oil content is the most important breeding goal inBrassica napus, and phenotyping is crucial to dissect its genetic basis in crops. To date, QTL mapping for oil content has been based on whole seeds, and the lipid distribution is far from uniform in different tissues of seeds inB. napus. In this case, the phenotype based on whole seeds was unable to sufficiently reveal the complex genetic characteristics of seed oil content.ResultsHere, the three-dimensional (3D) distribution of lipid was determined forB. napusseeds by magnetic resonance imaging (MRI) and 3D quantitative analysis, and ten novel oil content-related traits were obtained by subdividing the seeds. Based on a high-density genetic linkage map, 35 QTLs were identified for 4 tissues, the outer cotyledon (OC), inner cotyledon (IC), radicle (R) and seed coat (SC), which explained up to 13.76% of the phenotypic variation. Notably, 14 tissue-specific QTLs were reported for the first time, 7 of which were novel. Moreover, haplotype analysis showed that the favorable alleles for different seed tissues exhibited cumulative effects on oil content. Furthermore, tissue-specific transcriptomes revealed that more active energy and pyruvate metabolism influenced carbon flow in the IC, OC and R than in the SC at the early and middle seed development stages, thus affecting the distribution difference in oil content. Combining tissue-specific QTL mapping and transcriptomics, 86 important candidate genes associated with lipid metabolism were identified that underlie 19 unique QTLs, including the fatty acid synthesis rate-limiting enzyme-related geneCAC2, in the QTLs for OC and IC.ConclusionsThe present study provides further insight into the genetic basis of seed oil content at the tissue-specific level.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02324-0.