To understand the genetic diversity of Gobiopterus lacustris in the Leizhou Peninsula, transcriptomic sequencing of male and female gonads was performed. Using MISA software, 25 452 microsatellite markers were identified from the transcriptome data. A total of 14 708 single-base repeat types and 6 175 two-base repeat types were identified, while the numbers of three-, four-, five-, and six-base repeats were 4 223, 327, 15 and 4, respectively. By randomly selecting 50 microsatellite loci to design primers, 39 pairs of clear and stable bands could be amplified. The genetic diversity and genetic structure of G. lacustris were analyzed by microsatellite markers. It was showed that 11 microsatellite loci were polymorphic with different degrees of polymorphism, and the numbers of alleles ranged from 2 to 15, with an average number of 6 ± 3.9 (Table 2). The 11 microsatellite loci were used to analyze the genetic diversity of four populations including Zhanjiang Donghai Island population, Leizhou Jiulong Mountain population, Leizhou Fucheng population and Lianjiang Gaoqiao population. The average number of effective alleles of the four G. lacustris populations ranged from 1.919 to 2.485, and the observed heterozygosity and expected heterozygosity distributed at ranges of 0.343﹣0.465 and 0.381﹣0.483, respectively (Table 2). The average polymorphic information content (PIC) ranged from 0.348 to 0.465 (Table 2). Chi-square tests showed that most loci in the 4 G. lacustris groups did not deviate from Hardy-Weinberg equilibrium (P < 0.05) (Table 3). Linkage disequilibrium analysis was performed in each population and 18 pairs of loci were found to be significantly or very significantly (P < 0.05 or P < 0.01) deviated from linkage equilibrium. The genetic differentiation of the 4 populations reached above average level (0.107﹣0.216) (Table 5). Molecular variance analysis (AMOVA) revealed that genetic variation was mainly derived within the population (Table 6).