In this paper, Trionyx sinensis was used as research material, the difference of mitochondrial cytochrome b gene (Cytb) was compared and analyzed between Trionyx sinensis and Palea steindachneri or Pelodiscus axenaria. Cytb gene from HanShou Trionyx sinensis was cloned by using the PCR technology，and the expression patterns of different tissues and different growth stages were detected. Finally, the genetic relationship between Trionyx sinensis and Pelodiscus axenaria was used to analyze through molecular phylogenetic tree. It is found that the mRNA open reading frame of Cytb gene of Trionyx sinensis was found to contain 1 140 bp through cloning, encoding 379 amino acids. And the content of the bases for A + T (61.1%) was higher than that of G+C (38.9%). The amino acid sequential comparison with Palea steindachneri or Pelodiscus axenaria indicated that there were obvious differences. Moreover, Cytb gene was expressed in different tissues and different developmental stages of Trionyx sinensis. Besides, the highest expression was found in testis tissue. Furthermore, its expression level exhibited a “lowhighlow” pattern at different development stages. Further molecular evolutionary tree analysis suggested that Trionyx sinensis has a close relationship with Pelodiscus axenaria. However, there was distinguishable difference in Cytb between these two species. In addition, in view of the obvious difference in Cytb among mammals, fish and reptiles, Cytb  might play on important role in the evolution and genetic molecular marking in trionyx sinensis.