[Objectives] To investigate the genetic diversity and differentiation among populations of Gymnocypris chilianensis, Schizopygopsis pylzovi,and S. kialingensis distributedin Gansu Province, microsatellite markers were screened and used to assess the conservation genetics of these three species. [Methods]From 2017 to 2022, 309 ind from 16 populations of the three fish species were collected from the Weihe, Taohe, and Daxia Rivers (Yellow River basin); the Bailongjiang River (Jialing River in the Yangtze Valley); and the Shule, Heihe, and Shiyang Rivers (inland river systems). These included 110 ind from 5 populations of G. chilianensis, 72 ind from 6 populations of S. pylzovi, and 127 ind from 5 populations of S. kialingensis. High-throughput sequencing was conducted to identify microsatellite markers in the three species. Polymorphism at microsatellite loci was assessed using samples from the Schizothoracine species, and microsatellite polymorphism was detected by capillary fluorescence electrophoresis. GeneMaker 2.2.0 was used to read the capillary electrophoresis test results. Data were calibrated according to the base repeat unit at each SSR site to obtain genotyping data. Genetic diversity statistics were calculated using PopGene 1.32, including number of alleles (Na), effective number of alleles (Ne), Shannon’s index (I), Nei’s gene diversity (h), and percentage of polymorphic loci (P). Bayesian clustering analysis was performed using Structure software, with the length of the burn-in period parameter and the number of MCMC repeated sampling set to 1 × 10⁵ and 1 × 10⁶, respectively. The K values for the clustering group were set to 1﹣17, with each K value calculated 10 times. A UPGMA dendrogram was generated using Mega 7, with the bootstrap value set at 1 000. [Results] Capillary fluorescence electrophoresis identified 21 highly polymorphic primer pairs suitable for three species (Table 2). These 21 universal primer pairs were used to amplify 309 ind from 16 populations. Among the G. chilianensis populations, Huangyang population showed the highest genetic diversity (Na 1.442, Ne 1.314, I 0.203, h 0.203, P 44.24%), while Zhangye population had the lowest (Na 1.042, Ne 1.042, I 0.029, h 0.021, P 4.24%). For S. pylzovi, Qilian population had the highest genetic diversity (Na 1.539, Ne 1.193, I 0.206, h 0.127, P 53.94%), and Shimen population the lowest (Na 1.236, Ne 1.126, I 0.120, h 0.078, P 23.64%). S. kialingensis showed the highest genetic diversity in the Shimen population (Na 1.351, Ne 1.152, I 0.147, h 0.094, P 35.15%), with the lowest in the Zhouqu population (Na 1.181, Ne 1.089, I 0.088, h 0.057, P 18.18%) (Table 3). Overall, S. pylzovi exhibited higher genetic diversity than G. chilianensis and S. kialingensis. Genetic parameters indicated little intraspecific variation across different populations within species, suggesting comparable genetic diversity levels among populations. Bayesian genotype clustering detected interspecies gene flow between S. pylzovi and G. chilianensis in the Huangyang population, and between S. pylzovi and S. kialingensis in the Shimen population (Fig. 4). A UPGMA dendrogram of 16 Schizothoracine populations based on Nei’s genetic distance showed that S. pylzovi and S. kialingensis populationsformed small branches before clustering into a larger branch. The Shiyang population of S. pylzovi andall G. chilianensis populations formed one distinct branch (Fig. 5). [Conclusion] The genetic diversity of 16 populations across three Schizothoracine species was evaluated, thereby providing valuable insights into their germplasm status and supporting future conservation strategies.