Pseudorasbora parva was originally distributed in Jinshajiang and Nanpanjiang in Yunnan. However, it has been widely distributed in all major river systems and has become an invasive fish in other four river systems in Yunnan. In order to reveal the difference of genetic diversity and genetic differentiation of P. parva between Yunnan after the invasion and the native region, we collected 220 samples distributed in 13 different sites of Lancang River, Nujiang, Honghe and Irrawaddy River in Yunnan, and 6 native populations in Yellow River, Yangtze River, and Pearl River. Here we analysed a 1 072 base pair fragment of the mitochondrial cytochrome b gene (Cyt b) to examine the genetic diversity and genetic structure. A total of 72 mutation sites and 25 Cyt b haplotypes were identified. The haplotype diversity and nucleotide diversity of the four river systems in Yunnan were 0.828±0.014 and 0.005 44±0.001 18, respectively. Haplotype diversity of the introduced populations from the four river systems in Yunnan was higher than that of the native populations. The haplotype maximum likelihood tree and haplotype network map show that the haplotypes of the Yellow River population are special, while haplotypes in the invasive populations in Yunnan were mixed with those in Yangtze River and Pearl River. It is speculated that P. parva in Yunnan mainly came from the Yangtze River and the Pearl River, which was consistent with the history of introduction of economic fish in Yunnan Province. Analysis of molecular variance (AMOVA) revealed significant genetic subdivision among individuals within populations and among populations within rivers but not among rivers, indicating distribution of genetic diversity was inconsistent with contemporary hydrological structure. That may be the human effect who introduced them from many native regions. The results of Fu's Fs neutral test showed that there was no expansion of the invasive population of P. parva in Yunnan. In the subsequent management of the invasive population of P. parva in Yunnan, it is necessary to avoid the exchange of special haplotype groups with other regional groups, and to reduce the introduction of fish from different river systems. In addition, the development of new ways to utilize P. parva may be a useful way to control their population and reduce the damage to indigenous species and fishery culture in Yunnan.