Abstract: In this study we analyzed the genetic variations in the mtDNA D-Loop sequences of eight wild populations of grass carp (Ctenopharyngodon idella) including six populations (Hanjiang, Wujiang, Jiujiang, Shishou, Mudong, and Wanzhou) from the Yangtze River, the Zhaoqing population from the Pearl River, and the Nenjiang population from Heilongjiang River. A total of 34 variable sites and 34 haplotypes were detected in 424 individuals, and the haplotype diversity ranged from 0.474 to 0.708 among the eight populations. The pairwise population K2P genetic distances were between 0.0020 and 0.0049. The genetic distance between the three populations from the lower reach of the Yangtze River was the closest and their genetic differences were insignificant (P0.05). The Zhaoqing population also showed a close genetic distance with the three populations from the lower reach of the Yangtze River; and it did not have significant genetic differences compared to the Jiujiang population (P0.05). The Nenjiang population displayed a close genetic distance with the two populations from the upper reach of the Yangtze River. The genetic difference between the Nenjiang population and the Wanzhou population (P0.05) was insignificant. Furthermore we observed an extreme significant correlation between the genetic distance and the geographic distance (P0.01). The AMOVA analysis suggested that there were highly significant differences between populations (P0.01), and that the genetic variation among different watersheds in China accounted for 26.24% of the total variation. The haplotypes divided into two major branches which exhibited remarkable evolutionary differences (FST=0.644, P0.01), and this branching might originate in the late Pleistocene.