Buccinum striatissimum and Buccinum tenuissimum are commercially important whelks in the Sea of Japan. B. striatissimum is very similar to B. tenuissimum and their morphological variations make the identification of these species difficult, especially in case that these species are caught together because they are subject to the same environmental factors. In order to develop an easy and objective method to delineate these species, we utilized the difference of mitochondrial 16SrDNA of these two species. As a result, we could easily distinguish these two species by PCR-RFLP method using the difference of this region and AseI restriction endonuclease. In addition, we succeeded in distinguishing sympatric samples of these species, which suggests that these two species are likely to be reproductively isolated although the possibility of hybridization can not be completely ruled out.