Brachymystax lenok tsinlingensis (Salmonidae) is an indigenous and endangered cold-water fish in Tsinling mountain, China, and only is distributed as spot in the Weihe River and Hanjiang River among Tsinling mountain, To access relationship of populations from the two rivers and the genetic diversity of wild population, 11 microsatellite loci were firstly used to evaluate the genetic characterization using 120 samples from 2 tributaries (ZZ and LX populations) of the Weihe river and 2 tributaries (YX and TB population) of Hanjiang river. A total of 59 alleles were found and the number of alleles in each locus ranged from 3 to 8, with an average of 5.4. There were 6 highly polymorphic loci and 4 moderately polymorphic loci. The average expected heterozygosity (He) and observed (Ho) heterozygosity in four populations ranged from 0.6421-0.6980 and from 0.4841-0.6999. Significant deviations from Hardy–Weinberg were found in the four populations at some loci. Analysis of molecular variance (AMOVA) revealed that relatively little (11.02%) genetic diversity came from individual among population. In contrast, the majority of diversity (88.98%) occurred among individual within a population. Results from Nei’s genetic distance indicated that the YX and TB populations were grouped in one cluster, which was clustered with the ZZ population, the LX population was grouped in a separated cluster. Low genetic diversity and strong genetic differentiation have been found in this study. The baseline population genetic information supplied in this study will be vital for the protection and restoration of this endangered species.