Abstract: Qinling lenok (Brachymystax tsinlingensis Li), a national Class Ⅱ aquatic protected animal, is in urgent need of germplasm conservation due to the obvious decline of its wild population, however, since its subspecies was named in 1966, the status of its subspecies/species has been controversial until now. The unclear classification status seriously hinders the effective development of conservation work. Based on the mitochondrial control region gene sequence and microsatellite markers, we compared the molecular genetic differences between Qinling lenok and Heilongjiang lenok (Brachymystax lenok Pallas and Brachymystax tumensis Mori), to provide molecular evidence for clarifying the taxonomic status of Qinling lenok. The results showed as follows: (1) A total of 45 haplotypes were obtained by amplified mtDNA D-loop sequences of 217 samples, and no haplotypes were shared among above three groups. Phylogenetic tree based on haplotypes showed that each of the three groups of lenok was an independent clade. (2) The results of genetic differentiation based on 14 polymorphic loci showed that the genetic distance between Qinling lenok and B. lenok, Qinling lenok and B. tumensis was greater than the value between B. lenok and B. tumensis. (3) The genetic differentiation coefficient (F_ST) based on mitochondrial D-loop and polymorphic microsatellite loci analysis was higher than 0.25, indicating a high degree of genetic differentiation among the three groups. In the present study, a high degree of genetic differentiation among three groups were found. Combined with the previous results of obvious morphological differentiation between Qinling lenok and Heilongjiang lenok, which published by our team and the status of geographical isolation between Qinling and Heilongjiang rivers for a long time, we preliminarily determined that Qinling lenok is an independent species with Latin name Brachymystax tsinlingensis Li. It is suggested that Qinling lenok should be protected as an independent unit to avoid the destruction of germplasm resources caused by artificial introduction or hybridization.