Many transcriptional factors harboring the R2R3-MYB domain, basic helix-loop-helix domain, or WD40 repeats have been identified in various plant species as regulators of flavonoid biosynthesis in flowers, seeds, and fruits. However, the regulatory elements of flavonoid biosynthesis in underground organs have not yet been elucidated. We isolated the novel MYB genes IbMYB1 and IbMYB2s from purple-fleshed sweet potato (Ipomoea batatas L. Lam. cv Ayamurasaki). IbMYB1 was predominantly expressed in the purple flesh of tuberous roots but was not detected (or only scarcely) in other anthocyanin-containing tissues such as nontuberous roots, stems, leaves, or flowers. IbMYB1 was also expressed in the tuberous roots of other purple-fleshed cultivars but not in those of orange-, yellow-, or white-fleshed cultivars. Although the orange- or yellow-fleshed cultivars contained anthocyanins in the skins of their tuberous roots, we could not detect IbMYB1 transcripts in these tissues. These results suggest that IbMYB1 controls anthocyanin biosynthesis specifically in the flesh of tuberous roots. The results of transient and stable transformation experiments indicated that expression of IbMYB1 alone was sufficient for induction of all structural anthocyanin genes and anthocyanin accumulation in the flesh of tuberous roots, as well as in heterologous tissues or heterologous plant species.