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Flavonone 3-hydroxylase (F3H), anthocyanidin reductase (ANR) encoded by BANYULS (BAN) gene and glutathione S-transferase encoded by TRANSPARENT TESTA 19 (TT19) gene are three key enzymes in flavonoid pathway, playing important roles in the synthesis and transport of anthocyanin and proanthocyanidin. However, little is known about the expression variations and the regulatory network in Brassica. In this study, we examined their expression levels in seeds of 30 days after flowering (DAF) in F7 recombinant inbred lines (RILs) derived from a cross between Brassica nap us cultivars Youyan2 (black-seeded) and GH06 (yellow-seeded), and characterized the locus-level regulatory network related to B. nap us BAN, F3H and TT19 genes. Expression quantitative trait loci (eQTL) mapping revealed five, seven and eight eQTLs for BAN, F3H and TT19 genes, respectively. Marker E4M8 was associated with qBAN-8-2 and qTT19-8-2, while marker H022L18-2 was linked with qBAN-16-4 and qF3H-16-6, implying that there might be upstream regulatory genes in E4M8 and H022L18-2 marker flanking regions. In further analysis, we identified four trans-eQTLs (qBAN-16-5, qF3H-16-5, qBAN-16-4 and qF3H-11-2) close to the location of the major QTL controlling yellow-seeded trait of B. nap us. The 200-kb flanking sequences of the four eQTL on B. rapa chromosome A09 showed well synteny to B. oleracea genome sequence and partial continuous fragment of chromosome 1 of Arabidopsis genome, suitable for candidate gene predication of eQTLs. Based on gene finding results, seven transcription factors were suggested to be the potential upstream candidate(s) controlling expression variations of BAN, F3H and TT19 genes. These results could provide a new approach for constructing regulatory pathways that contribute to complex traits, such as yellow-seeded trait.