An analysis of eight microsatellite loci in 90 individuals was performed to define the genetic structure and variability of three estuarine populations of Coilia mystus: 30 individuals each collected from ChangJiang River (Yangtze River) estuary-CJ, MinJiang River estuary-MJ, and ZhuJiang River (Pearl River) estuary-ZJ. The results showed polymorphic information contents (PIC) of 0.78, 0.77, and 0.64 in CJ, MJ, and ZJ, respectively. The mean observed number of alleles (Na) ranged from 7.38 to 11.88. The mean observed and expected heterozygosities ranged from 0.09 to 0.21 and from 0.68 to 0.81, respectively. The ZJ population was least polymorphic. Highly significant deviations from Hardy-Weinberg equilibrium, mostly due to deficits of heterozygote, were found in these three populations. Pairwise FST and Reynolds’ distance indicated that the three estuarine populations were genetically distinct, in accordance with the principal component analysis (PCA) and the Bayesian model-based clustering algorithm analysis. The results showed that pairwise genetic differentiation among these three estuarine populations were relatively high, with possible divergence to subspecies level. This study provides microsatellite DNA evidence for assessing the genetic distinctness of C. mystus populations and will benefit fishery resource management and sustainable utilization of C. mystus.