To assess the genetic diversity and genetic variation of Coilia brachygnathus populations in the mid-lower Yangtze River, three geographical populations from Dongting Lake, Changhu and Chaohu were studied based on mitochondrial D-loop sequences. Some genetic diversity parameters were analyzed by DnaSP 5.0, and heterozygous molecular variation analysis (AMOVA) was acquired by Arlequin 3.1.1. Using MEGA5.0, we analyzed the base composition, the number of nucleic acid site replacement, the calculation of the genetic distance between populations and among populations. We also constructed the neighbor joining (NJ) phylogenetic tree, and the NJ system tree based on the topology of each branch. Network 5.1 was used to analyze the sequence of the control zone, and then the haplotype network diagram was constructed. Genetic diversity and genetic structure of C. brachygnathus populations in the mid-lower Yangtze River were analyzed. The results showed that there were 1 236 bp, 90 variation sites and 54 parsimony informative sites in D-loop sequence. There were 58 haplotypes among three geographical populations in the mid-lower Yangtze River, and the haplotypes diversity range was 0.949 ~ 0.982, the diversity of nucleotides was in the range of 0.004 99 ~ 0.006 21, indicating that the three geographical groups in the mid-lower Yangtze River had a high genetic diversity level (Table 2, 3). The genetic differential index Fst of total for populations was 0.265 95 (Table 4), showing that the genetic differentiation among populations was moderate and significant, especially between the Chaohu population and other groups. Similar results were also obtained according to the NJ phylogenetic tree and haplotype network diagram (Fig. 1, 2).