Radish with red skin and red flesh (Raphanus sativus) is a unique vegetable containing large amounts of a red pigment, which is widely used in foods, wine, and cosmetics. To investigate the gene or genes that play a key role in anthocyanin biosynthesis in radish with red skin and red flesh, the red pigment content and expression of genes involved in anthocyanin biosynthesis of 16 varieties with different colored flesh were studied. The expression level of the genes RsPAL, RsCHS, RsCHI, RsDFR, RsF3H, RsF3'H, and RsANS in radish with red skin and red flesh are all significantly higher than that of radish with white skin and white flesh, radish with red skin and white flesh, radish with green skin and pinky flesh, and radish with red skin and pinky flesh. Correlation analysis indicated that the gene expression level of RsDFR, RsF3H, RsCHS, RsANS, RsF3'H, RsCHI, and RsPAL showed remarkable positive and significant correlation to red pigment content of radish. Stepwise regression analysis showed that the gene expression level of RsDFR had the highest and significant direct effect (0.8932) on red pigment content of radish. The results indicated that (1) the red pigment content of radish is closely related to the increased expression of a number of structural genes in anthocyanin biosynthesis, (2) the RsDFR gene plays a key role in anthocyanin biosynthesis in red radish with red flesh, and (3) RsDFR might be one of the best targets in genetic engineering for anthocyanin production from radish and other plants.