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Anthcyanins determine the colors of flowers, fruits, and purple vegetables and act as important health-promoting antioxidants. BT 126 represents a broccoli variety with a high content of anthocyanins (5.72 mg/g FW). Through QTL-seq bulk segregant analysis, the present study aimed to determine the quantitative trait loci (QTLs) involved in anthocyanin biosynthesis in the F2 population (n = 302), which was obtained by crossing BT 126 with a non-anthocyanin-containing SN 60. The whole-genome resequencing of purple (n = 30) and green (n = 30) bulk segregates detected ~1,117,709 single nucleotide polymorphisms (SNPs) in the <i>B. oleracea</i> genome. Two QTLs, tightly correlated with anthocyanin biosynthesis (<i>p</i> < 0.05), were detected on chromosomes 7 (<i>BoPur7.1</i>) and 9 (<i>BoPur9.1</i>). The subsequent high-resolution mapping of <i>BoPur9.1</i> in the F2 population (n = 280) and F3 population (n = 580), with high-throughput genotyping of SNPs technology, narrowed the major anthocyanin biosynthesis QTL region to a physical distance of 73 kb, containing 14 genes. Among these genes, <i>Bo9g174880</i>, <i>Bo9g174890,</i> and <i>Bo9g174900</i> showed high homology with <i>AT5G07990</i> (gene encoding flavonoid 3′ hydroxylase), which was identified as a candidate gene for <i>BoPur9.1</i>. The expression of <i>BoF3’H</i> in BT 126 was significantly higher than that in SN60. Multiple biomarkers, related to these QTLs, represented potential targets of marker-assisted selection (MAS) foranthocyanin biosynthesis in broccoli. The present study provided genetic insights into the development of novel crop varieties with augmented health-promoting features and improved appearance.