Abstract: To evaluate the genetic diversity and genetic structure of grass carp (Ctenopharyngodon idella), we analyzed the genetic variation of mitochondrial DNA D-loop region among four wild populations (Hanjiang, Jiujiang, Shishou, Wujiang) and two domesticated populations after two generations (F1 and F2) of selective breeding. The results showed that the wild populations had higher number of haplotype (H), haplotype diversity (H_d), nucleotide diversity (π) and average number of nucleotide differences (K) compared with the domesticated populations. Between the bred populations, F1 generation was higher than F2 generation in the aspects of nucleotide diversity (π) and average number of nucleotide differences (K), but haplotype diversity (H_d) in F1 generation was lower than F2 generation. Haplotype analysis showed that all six populations did not share a haplotype. By contrast, the four wild populations shared two haplotypes (Hap1 and Hap3). The Shishou population and two domesticated populations shared one haplotype (Hap15). Genetic differentiation index (F_st) analysis showed that there was great genetic differences between wild (Hanjiang, Jiujiang, Wujiang) and domesticated populations (range F_st from were 0.41475 to 0.55128). Genetically, Shishou population was closely related with F1 population, but its relationship with F2 was only moderate. Moreover, the genetic differentiation level between two breeding populations was small (F_st=0.05741). The analysis of neighbor-joining phylogenetic trees based on 276 individuals among six populations and haplotype network graph based on 27 haplotypes indicated the wild populations (Hanjiang, Jiujiang, Wujiang) had a distant relationship with those domesticated populations. Although Shishou population and two breeding populations were more closely related. The above results showed that among the two domesticated populations, the genetic structure has already changed after two generations of selective breeding, and the genetic diversity also decreased with the development of the breeding program. The results also urge us that the current breeding strategy should be improved in order to maintain the genetic diversity of the breeding populations and reduce the risk of inbreeding.