Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in-depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47a^ZY821, but not the yellow-seed allele BnA09MYB47a^GH06, promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops.