Background: DNA barcoding is an effective method of accurately identifying morphologically similar oyster species. However, for some of Japan's Ostrea species there are no molecular data in the international DNA databases. Methods: We sequenced the mitochondrial large subunit ribosomal DNA (LSrDNA) and cytochrome c oxidase subunit I (COI) gene of five known and two unidentified Ostrea species. Phylogenetic comparison with known Ostrea species permitted accurate species identification by DNA barcoding. Results: The molecular data, which were deposited in an international DNA database, allowed for a clear distinction among native Ostrea species in Japan. Moreover, the nucleotide sequence data confirmed that O. stentina (Atsuhime-gaki) inhabits Kemi and Ibusuki, Japan. Conclusions: This is the first record of O. stentina in Japan. These results provided for accurate species identification by DNA barcoding of the taxonomically problematic species O. futamiensis, O. fluctigera, O. setoensis and O. stentina in Japan.