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Abstract Oxygymnocypris stewartii is a cold‐water cyprinid indigenous to China, where it inhabits the Yarlung Zangbo river and its tributaries at high elevations on the Qinghai–Tibet plateau. Owing to its restricted distribution and a gradual reduction in population size, this fish is listed as near threatened on the IUCN Red List, and it is classified as endangered at the national level. To investigate the species’ genetic diversity, this study used 26 pairs of microsatellite markers to analyse the genetic structure of six geographic populations of O. stewartii in the Yarlung Zangbo river basin (YZRB). The results showed moderate genetic diversity in O. stewartii, with an average effective number of alleles of 2.4052, expected heterozygosity of 0.4759, and polymorphism information content of 0.4210. Genetic structure analysis detected significant differentiation among the six populations (p