Abstract: The primers for Pelodiscus sinensis and Pelodiscus axenaria were designed by using the sequences of mitochondrial DNA 12S rRNA region of Dogania subplana , Chrysemys picta and Chelonia mydas , 562 base pairs of mitochondrial DNA were amplified and sequenced. Theresultsshowthatthelengthoftheirsequenceisthesame,theA,T,G,Ccontentsaresimilar,thenumberofA,T,G, Cis 209 (37.2%) ,121 (21.5%) ,145 (25.8%) ,87 (15.5%)in Pelodiscus sinensis and 207 (36.8%),120 (21.4%),145 (25.8%), 90 (16 %)in Pelodiscus axenaria. Based on their sequence data , we found there are 13 different nucleotide sites (percentage divergence is 2. 31 %) between Pelodiscus sinensis and Pelodiscus axenaria by using the software of ClustalW , whereas the average number of different nucleotide sites is 3 (percentage divergence is 0. 53 %) in Pelodiscus sinensis and 2 (percentage divergence is 0. 36 %) in Pelodiscus axenaria. The nucleotide percentage divergence is obvious between species , so it suggests Pelodiscus axenaria is a new species in Trionyx. Restriction endonuclease analysis based on sequence data of this DNA fragment revealed the presence of polymorphic sites for MspI endonuclease , there is one site for MspI endonuclease in Pelodiscus axenaria , but none in Pelodiscus sinensis. The restriction profiles obtained by agarose gel electrophoresis when amplicons were cut with MspI enzyme allowed the unequivocal identification of Pelodiscus sinensis and Pelodiscus axenaria.