Genetic diversity is an important component of biodiversity and investigating and protecting the wild genetic diversity of species has always been an important research topic in conservation biology. Due to habitat destruction and over catching, wild Chinese soft-shell turtles (Pelodiscus spp.) have been severely damaged, resulting in the species being listed on the IUCN Red List of Threatened Species in 2000. However, only few studies with contradictory results on the genetic diversity of Pelodiscus turtles have been reported. To investigate the genetic diversity and population structure of Pelodiscus turtles, 123 specimens were collected from five localities in East Asia, and their genetic variation was analyzed on the basis of a 922-bp partial sequence of the mitochondrial cytb gene. Forty-nine polymorphic sites were detected, revealing 56 haplotypes. A pattern of high haplotype diversity (Hd = 0.994) and nucleotide diversity (π = 0.01655) was found in the examined range. A maximum likelihood phylogenetic tree and median-joining network analyses indicated that the Pelodiscus turtles divided in four populations throughout East Asia, and the Pelodiscus turtles probably originate from the Yangtze River and was introduced to Yellow River, Taiwan, and Japan through Xijiang River. These results served a helpful resource for conservation of Pelodiscus turtles.