The skin color of grape berry is very important in the wine industry. The red color results from the synthesis and accumulation of anthocyanins, which is regulated by transcription factors belonging to the MYB family. The transcription factors that activate the anthocyanin biosynthetic genes have been isolated in model plants. However, the genetic basis of color variation is species-specific and its understanding is relevant in many crop species. This study reports the isolation of , and genes from muscadine grapes for the first time. They are designated as (GenBank Accession No. KJ513437), and ( ) (GenBank Accession No. KJ513438). The findings in this study indicate that, the deduced and protein structures share extensive sequence similarity with previously characterized plant MYBs, while phylogenetic analysis confirms that they are members of the plant MYB super-family. The expressions of , and ( ) gene sequences were investigated by quantitative real-time PCR using in vitro cell cultures, and berry skin samples at different developmental stages. Results showed that , and genes were up-regulated in the veràison and physiologically mature red berry skins during fruit development, as well as in in vitro red cell cultures. This study also found that in ripening berries, the transcription of , and in the berry skin was positively correlated with anthocyanin accumulation. Therefore, the upregulation of , and results in the accumulation and regulation of anthocyanin biosynthesis in berry development of muscadine grapes. This work greatly enhances the understanding of anthocyanin biosynthesis in muscadine grapes and will facilitate future genetic modification of the antioxidants in .