To clarify the genetic structure and evolutionary characters in three populations of Coilia mystus, the mitochondrial DNA cyt b gene was used in this study. A total of 65 fish, i.e., 21 individuals from the Yangtze River estuary, 22 from the Minjiang River estuary, and 22 from the Pearl River estuary, were collected. Sequences of cyt b gene segments amplified by PCR were used. 607 consensus sequences of cyt b were obtained. Thereinto, 102 were variable sites, accounting for 16.8% of total sequence, and 68 were parsimony sites. They were all transitional and transversional variations, and no indels were found. The content of A+T(57.6%) is bigger than G+C(42.4%). The index of nucleotide diversity is largest in the Minjiang population among these three populations. Selection tests disclose that natural selection operates at molecular level within these three populations.In the total 65 sequences, 34 distinct haplotypes were detected. Genetic distance within the populations were between 0.3% and 1.2%, and between populations were from 0.8% to 10.8%. The largest genetic distance was 10.8% between the Yangzte River population and the Pearl River population, and the smallest was 0.8% between the Minjiang River population and the Pearl River population. AMOVA analysis disclosed that variation between populations accounting for 90.25% of total variation,suggesting between population variations are the main source of total variation. This study suggests that the genetic divergence between the Yangtze River population and the Minjiang River population, the Pearl River population maybe arrived at subspecies level.