The Zig-zag eel (Mastacembelus armatus) is an economically important species in southern China. Its natural resources have declined year by year due to overfishing. Understanding its genetic diversity and population structure is very important for resource conservations. Here, we first successfully developed 28 polymorphic microsatellite markers for zig-zag eels and ten of them were used to examine the genetic diversity and differentiation of 7 populations collected from the major river systems of south China. In total, 224 alleles were found with the 10 microsatellite loci in 7 populations, ranging from 4.6 (Nandujiang: NDJ) to 11.1 (Xijiang, XJ), with an average of 8.871 alleles. The average observed and expected heterozygosity ranged from 0.550 (NDJ) to 0.964 (Yuangjiang, YJ) and from 0.537 (NDJ) to 0.775 (Tanjiang, TJ), respectively. The average polymorphism-information content ranged from 0.472 (NDJ) to 0.757 (TJ). Hardy–Weinberg equilibrium test results revealed the loci showed differing deviation in different populations. In total, low level of genetic diversity was only found in HJ (Hanjiang) and NDJ populations. Besides, evidence of recent bottleneck was found in the HJ populations. Analysis of molecular variation showed that the percent variation within individuals (75.00%) was higher than that among populations (25%). In addition, population structure and the pairwise FST revealed that there was low differentiation among XJ, TJ and YJ populations. These data provide important genetic resources for understanding the population differentiation and facilitating genetic conservation and utilization of this species.