Abstract: Mitochondrial 16S rRNA gene fragments were amplified from Pinctada fucata, P.chemnitzi and Pteria penguin using PCR technique. The PCR products were directly sequenced, and 439 bp nucleotide sequences were obtained after excluding the primers and truncating partial sequences at the end. Sequence variation was analyzed using MEGA 3.1 software. The results indicated that base substitution was not found among P.fucata, but two transversions were found among P.chemnitzi, one transition and five transversions among P.penguin. Alignment with nine homologous sequences from Pteridae species in GenBank revealed 51 insertion/deletions and 231 variable sites (173 parsimony-informative sites and 53 singletons) among 464 truncated alignment sites. The percentage of the identity of the 16S rRNA gene fragments was 83.2% between P.fucata and P.chemnitzi, 55.4% between P.fucata and Pteria penguin, and 59% between P.chemnitzi and P.penguin. The NJ tree indicated that P.fucata and P.chemnitzi were assembled together with Pinctada species, yet P.penguin assembled together with the Pteria species, which was consistent with the morphological classification.