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Anthocyanins are well-known antioxidants that are beneficial for plants and consumers. Dihydroflavonol-4-reductase (<i>DFR</i>) is a key gene of anthocyanin biosynthesis, controlled by multiple transcription factors. Its expression can be enhanced by mutations in the negative regulator of anthocyanin biosynthesis myeloblastosis family transcription factor-like 2 (<i>MYBL2</i>). The expression profiles of the <i>DFR</i> gene were examined in 43 purple and green varieties of <i>Brassica oleracea</i> L., <i>Brassica napus</i> L., <i>Brassica juncea</i> L., and <i>Brassica rapa</i> L. <i>MYBL2</i> gene expression was significantly reduced in purple varieties of <i>B. oleracea</i>, and green varieties of <i>B. juncea</i>. The <i>MYBL2</i> gene sequences were screened for mutations that can affect pigmentation. Expression of the <i>DFR</i> gene was cultivar-specific, but in general it correlated with anthocyanin content and was higher in purple plants. Two single nucleotide polymorphysms (SNPs) were found at the beginning of the DNA-binding domain of <i>MYBL2</i> gene in all purple varieties of <i>B. oleracea</i>. This mutation, leading to an amino acid substitution and the formation of a mononucleotide repeat (A)₈, significantly affects RNA structure. No other noteworthy mutations were found in the <i>MYBL2</i> gene in green varieties of <i>B. oleracea</i> and other studied species. These results bring new insights into the regulation of anthocyanin biosynthesis in genus <i>Brassica</i> and provide opportunities for generation of new purple varieties with precise mutations introduced via genetic engineering and CRISPR/Cas.