BackgroundSeed oil content is an important agronomic trait ofBrassica napus(B. napus), and metabolites are considered as the bridge between genotype and phenotype for physical traits.ResultsUsing a widely targeted metabolomics analysis in a natural population of 388B. napusinbred lines, we quantify 2172 metabolites in mature seeds by liquid chromatography mass spectrometry, in which 131 marker metabolites are identified to be correlated with seed oil content. These metabolites are then selected for further metabolite genome-wide association study and metabolite transcriptome-wide association study. Combined with weighted correlation network analysis, we construct a triple relationship network, which includes 21,000 edges and 4384 nodes among metabolites, metabolite quantitative trait loci, genes, and co-expression modules. We validate the function of BnaA03.TT4, BnaC02.TT4, and BnaC05.UK, three candidate genes predicted by multi-omics analysis, which show significant impacts on seed oil content through regulating flavonoid metabolism inB. napus.ConclusionsThis study demonstrates the advantage of utilizing marker metabolites integrated with multi-omics analysis to dissect the genetic basis of agronomic traits in crops.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-023-02984-z.