Dihydroflavonol reductase is a key enzyme in anthocyanidin synthesis of plants. Cloning and analysis of Dihydroflavonol reductase gene (DFR) was carried out in China’s peculiar radish germplasm ‘Xinlimei’ with red flesh. By blasting the complete CDS sequence of Chinese cabbage DFR gene with white radish inbred line ‘36-2’ whole genome sequences (unpublished), a homologous single gene Rsa10008592 was obtained. The complete CDS of RsDFR was amplified from the fleshy root of ‘Xinlimei’ radish inbred line ‘HX12Q-49’ with the primers designed according to the Rsa10008592 sequence by RT-PCR. The ORF of RsDFR contained 1,164 bp (Genbank accession number: KF280272), which encoded the protein of 384 aa. The sequence difference of 19 nucloetide acids and 3 amino acids were found between ‘Xinlimei’ radish and white radish through sequences comparison. Phylogenetic tree showed RsDFR had the highest homology with the DFRs of Chinese cabbage and mustard. qRT-PCR result showed the expression patterns of the RsDFRs in the fleshy roots of ‘Xinlimei’ radish and white radish were different. RsDFR in white radish only expressed at early developmental stage, while it expressed at all developmental stages and reached the peak at the splitting stage in ‘Xinlimei’ radish. The protein from RsDFR was further analyzed.