The silver carp (), one of the four major Chinese carps, is widely distributed in the Yangtze, Pearl, and Heilongjiang River Basins of China. The wild silver carp populations in the Yangtze River, with an excellent production performance, is an important germplasm resource for artificial farming and breeding. Because of overfishing, habitat fragmentation, and water pollution, the natural carp populations have been continuously declining in recent years. The basic genetic background analysis of the silver carp would be theoretically and practically important for the conservation of germplasm resources and genetic improvement. In this study, mitochondrial cytochrome C oxidase subunit I gene (COI) was sequenced to determine the genetic diversity and variation of six geographical silver carp populations:Yibin, Zhongxian, Wanzhou, Shishou, Jianli, and Xiangjiang. The amplified fragment was 648 bp in length, and a total of 26 haplotypes were defined among 123 sequences of COI from the six silver carp populations. The haplotype and nucleotide diversity index of the six populations ranged from 0.0476 to 0.0945, and from 0.00196 to 0.00982, respectively. The six groups exhibited a high genetic diversity, of which the Wanzhou population showed the highest haplotype and nucleotide diversities; the lowest haplotype and nucleotide diversities were observed in the Zhongxian and Shishou populations, respectively. The molecular variance (AMOVA) analysis showed that the variation within populations (accounting for 88.72%) was the main source of variation, and only 11.28% of the variation was found among the groups. The haplotype network was radial, centering on the main haplotype. The neighbor-joining phylogenetic tree of haplotypes based on genetic distances were inconsistent with their geographical distances. According to the differentiation index (, there were high differences between the Yinbin, Wanzhou, and 3 populations from the middle reach ( < 0.05); meanwhile, the Jianli population showed a significant differentiation from the Shishou and Xiangjiang populations ( < 0.01). The results revealed that the upper-and middle-reach populations should have originated from different populations in the Yangtze River. This study could provide useful basic data for the scientific protection, and reasonable and sustainable utilization of this germplasm resource in the Yangtze River Basin.