To comprehend the genetic diversity within the Chinese soft-shelled turtle(Pelodiscus sinensis), mitochondrial genes(Cyt b) from two geographical populations of P. sinensis and their hybrids were sequenced and analyzed. The study yielded a total of 90 sequences with 18 haplotypes among the three sequenced groups of Japanese population, Huangsha population and hybrid population of P. sinensis via PCR. Among the 90 individuals of Cyt b, the average base contents for A, T, C and G was 35.4%, 26.0%, 27.9% and 10.7%, respectively, revealing a distinct anti-G bias. The Japanese population, Huangsha population and hybrid population exhibited 5, 6 and 10 haplotypes, respectively. Notably, the Japanese population shared haplotypes Hap 8, Hap 9 and Hap 16 with the hybrid population. Population variance contributed significantly to overall variation at 60.8%. Haplotype diversity for all three populations ranged from 0.747 to 0.867, average nucleotide differences ranged from 12.922 to 16.262, and nucleotide diversity ranged from 0.009 13 to 0.011 51. In terms of the three groups, the number of haplotypes, haplotype diversity, average nucleotide diversity and nucleotide diversity of the hybrid population were the highest, and those of the Japanese population were the lowwest, while the number of polymorphic loci decreased in the order of the Huangsha population, hybrid population and Japanese population. The study findings underscored the rich genetic diversity of hybrid population of P. sinensis, suggesting that hybridization events augment the genetic diversity within P. sinensis.