Among several genera of the family Ostreidae, Ostrea, Crassostrea and Saccostrea play a central role in the near shore brackish ecosystem worldwide. Although 9 species of oysters belonging to these 3 genera had been found in Kyushu, Japan, oysters are highly variable in form with ecomorphological variations, and molecular methods can usefully complement morphological in determining the status of oyster taxa. In this study, a simple DNA fi ngerprinting analysis of the mitochondrial DNA segment encoding the 16S ribosomal RNA gene was developed for differentiation of 3 genera oysters. Of 2 Ostrea, 4 Crassostrea, and 2 Saccostrea species distributed in Kyushu, Japan, nucleotide sequences of the mitochondrial DNA 16S ribosomal RNA gene were aligned to detect unique sites of restriction enzymes for genus and species identifi cations by means of PCR-RFLP analysis. PCR amplifi cation of an apparent 530 bp fragment of the partial 16S ribosomal RNA gene was successfully performed on all specimens collected. Direct double digestion of the PCR products using Eco130I and HindIII enzymes enabled to discriminate Ostrea, Crassostrea, and Saccostrea genera. In addition, double and single digestions of the PCR products using DdeI and AluI enzymes and AluI enzyme alone enabled to identify 4 Crassostrea species and 3 Saccostrea species, respectively.