Abstract: In this study, the genetic structure of Gambusia affinis which come from 8 different geographical populations in Yunnan was analyzed using mitochondrial cytochrome b technology. 786 bp sequences were obtained from 210 individuals of G.affinis, collected from 8 geographic populations in Yunnan Province. Of the 786 bp sequences, 5 polymorphic sites defined 5 haplotypes. Among the haplotypes, the haplotype H1 was shared by 156 samples from 7 geographic populations (74.3% of the total length), and the haplotype H2 was shared by 52 samples which come from 7 geographic populations (24.8% of the total length). The base frequency content of G.affinis (A + T) was 53.2%, which was greater than (G + C) 46.8%. The nucleotide polymorphisms were the highest in the Dongdahe River population and the remaining 7 populations were lower. The gene flow between populations was 0.28. The results of molecular variance analysis (AMOVA) showed that 52.74% variation derived from different populations, 47.26% of the variation existed within the same population. The variation among the populations was the main source of total variation. The neutral analysis of the population of G.affinis showed that the population of G.affinis in Yunnan was relatively stable.