Abstract: The primers for Pelodiscus sinensis, Pelodiscus axenaria and Palea steindachneri were designed by using the sequences of mitochondrial DNA tRNA2Glu, tRNA2Thr gene of Dogania subplana, Chrysemys picta and Chelonia mydas, 1140 base pairs of mitochondrial DNA cytb gene were amplified and sequenced.The results show that the length of their sequences is the same, A, C, G, T contents are similar, the number of A, C, G, T is 378 (33.2%),322 (28.2%),122 (10.7%),318 (27.9%-in Pelodiscus sinensis: 373(32.7%),324(28.4%),124(10.9%),319 (28.0%-in Pelodiscus axenaria and 380 (33.3%),330 (28.9%),127(11.1%),303(26.7%-in Palea steindachneri.Based on their sequence data, analysis with the software of Jellyfish, we can know: the nucleotide percentage homology is 92.3% between Pelodiscus sinensis and Pelodiscus axenaria, 85.0% between Pelodiscus sinensis and Palea steindachneri, 84.1% between Pelodiscus axenaria and Palea steindachneri; the nucleotide percentage divergence is 7.7% between Pelodiscus sinensis and Pelodiscus axenaria, 15.0% between Pelodiscus sinensis and Palea steindachneri, 15.9% between Pelodiscus axenaria and Palea steindachneri, whereas the individual nu-cleotide percentage divergence is 2.37% in Pelodiscus sinensis, 0.88% in Pelodiscus axenaria and 0.18% in Palea stein2 dachneri.Restriction endonuclease analysis based on sequence data of their DNAfragments revealed the presence of polymorphic sites for Nde Ⅰand BamH Ⅰendonucleases.The restriction profiles obtained by agarose gel electrophoresis when amplicons were respectively cut with Nde Ⅰand BamH Ⅰenzymes allowed the unequivocal molecular identification of Pelodiscus axenaria, Palea steindachneri and Pelodiscus sinensis.Based on their obvious nucleotide percentage divergence and the changes of poly2 morphic sites for Nde Ⅰand BamH Ⅰendonucleases in their sequences, it from the molecular level suggests Pelodiscus axe2 naria is a new species in Pelodiscus.