Carassius auratus in Pu'an and Caohai Lake are the special isolated groups of C. auratus in the Karst Mountain Areas. In order to fully understand the genetic structure systematically and discuss the phylogenetic relationship and genetic differentiation of the two populations, the mitochondrial DNA control regions of two geographical populations were analyzed by sequencing technology in this study. The results showed that C. auratus in Pu'an had eight types of sequence length of 919 and 921-927 bp, belonging to 27 kinds of genotypes and 14 kinds of haplotypes; C. auratus in Caohai Lake had three sequence types of sequence length of 923-925 bp, belonging to 11 kinds of genotypes and 6 kinds of haplotypes; The sequences variation due to base substitution and the deletion/insertion. Clustering analysis confirmed that C. auratus in Pu'an were the mixed groups of C. auratus gibelio and C. auratus including their 3 sublineages. C. auratus in Caohai Lake was the mixed groups of 2 sublineages of C. auratus. The populations of C. auratus and C. auratus in Pu'an had medium genetic diversity and the populations of C. auratus and C. auratus in Caohai Lake had low genetic diversity, while the population of C. auratus gibelio in Pu'an had extremely low genetic diversity. The study indicated that the two geographical groups of C. auratus had a characteristic of complicated group composition and matrilineal diversity. As the longterm geographical isolation, reproductive isolation and lack of gene flow resulted in the difference of genetic drift and evolutionary rate, which made the genotype and haplotype of D-loop also had obvious geographical distribution pattern, the genetic differentiation was remarkable among various groups. C. auratus gibelio in Pu'an and in northeast of China belong to the same origin of matrilineal line, the degree of population genetic variation was so low that improving the quality of the parents was urgent. The founder effect, the bottleneck effect and genetic drift led to the low genetic diversity current situation of C. auratus in Caohai Lake. This experiment might enrich the fish population genetics research materials in the Karst Mountain and provide basic data for identification and evaluation of the germplasm resources of the local C. auratus.