| Summary: | Dihydroflavonol 4-reductase (<i>DFR</i>) is a key enzyme in the flavonoid biosynthetic pathway and is essential for the formation of plants’ color. In this study, 26 <i>BnDFR</i> genes were identified using 6 <i>Arabidopsis DFR</i> genes as reference. The physicochemical properties, subcellular localization, and conserved structure of <i>BnDFR</i> proteins were analyzed; the evolutionary relationship, collinearity analysis, and expression characteristics of <i>BnDFR</i> genes were studied; and the correlation between the expression level of <i>BnDFR</i> genes and anthocyanin content in rape petals were analyzed. The results showed that the 26 <i>BnDFRs</i> were located in chloroplasts, cytoplasm, nuclei, and mitochondria, distributed on 17 chromosomes, and divided into 4 groups; members of the same group have a similar function, which may be related to the environmental response elements and plant hormone response elements. Intraspecific collinearity analysis showed 51 pairs of collinear genes, and interspecific collinearity analysis showed 30 pairs of collinear genes. Analysis of the expression levels of <i>BnDFRs</i> and anthocyanin content in different color rape petals showed that <i>BnDFR6</i> and <i>BnDFR26</i> might play an important role in the synthesis of anthocyanins in rape petals. This provides theoretical guidance for further analysis of the anthocyanin anabolism mechanism involved in the <i>DFR</i> gene in <i>Brassica napus</i>.
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