[Objectives] Microsatellite markers have been widely used in the research of conservation genetics of aquatic animals. However, there is no report on microsatellite loci of Gymnodiptychus pachycheilus. [Methods] From 2014 to 2022, a total of 2 G. pachycheilus populations (56 individuals) were collected from the main stream of the middle Yalong River and Weihe River. Illumina HiSeq sequencing was used to explore microsatellite markers from the genome of G. pachycheilus. Microsatellite loci were tested using 2 wild population samples’ DNA of G. pachycheilus. Null alleles were tested using the software MICRO-CHECKER version 2.2.3, genetic diversity statistics were obtained using AUTOTET. Deviations from Hardy-Weinberg equilibrium and linkage disequilibrium at each locus were calculated using GENEPOP 4.7.5, and significance values were adjusted after applying Bonferroni correction. Polymorphism information content (PIC) value was based on PIC_Calc0.6. [Results] 567 200 microsatellite loci were identified from 85 905 individual sequence reads produced, and 15 primer pairs were designed and testified in the Yalong River Xinlong population and the Weihe River population. In the Xinlong population, the average number of observed alleles per locus was 6.87 (from 3 to 13), the average observed heterozygosities and PIC were 0.712 4 and 0.630 3 respectively, 1 pair linkage disequilibrium was detected and eight loci showed significant deviation from Hardy-Weinberg equilibrium among the loci (Table 2). In the Weihe population, the average number of observed alleles per locus was 8.4 (from 4 to 16), the average observed heterozygosities and PIC were 0.719 5 and 0.680 7 respectively, 4 pairs linkage disequilibrium was detected and 7 loci showed significant deviation from Hardy-Weinberg equilibrium among the loci (Table 3). [Conclusion] These 15 high polymorphism microsatellite loci described for G. pachycheilus will be useful for future study on phylogeography and genetic diversity studies.