In recent years, the influence of historical geological and climatic events on the evolution of flora and fauna in the Tibetan Plateau has been a hot research topic. The Qilian Mountain region is one of the most important sources of biodiversity on the Qinghai-Tibet Plateau. Many species existed in the region during the Pleistocene glacial oscillation, and the complex geographical environment provided suitable conditions for the survival of local species. The shrinkage, expansion, and transfer of the distribution range and population size of species have significant effects on genetic diversity and intraspecific differentiation. To reveal the effects of geological uplift and climate oscillation on the evolution of fish populations in the Qilian Mountains, we investigated the genetic structure, phylogenetic relationship, and phylogeographical characteristics of genus Triplophysa species in the Qilian Mountains using the mitochondrial DNA gene (COI), three nuclear genes (RAG1, sRH, and Myh6) and 11 pairs of nuclear microsatellite markers. We collected 11 species of genus Triplophysa living in the Qilian Mountains, among which Triplophysa hsutschouensis and Triplophysa papillosolabiata are widely distributed in the rivers on the northern slope of the Qilian Mountains.There was a high degree of lineage differentiation among species, and the genetic diversity of endemic species was low. The different geographical groups of T. papillosolabiata presented some allogeneic adaptation and differentiation, which was closely related to the changes in the river system. Except for the population expansion event of T. hsutschouensis during the last glacial period of the Qinghai-Tibet Plateau (0.025 MYA), the population sizes of other plateau loach species remained stable without significant population expansion. Starting from the east and west sides of the Qilian Mountains, T. hsutschouensis, and T. papillosolabiata showed two species colonization routes in opposite directions. The geological events of the uplift of the Qinghai-Tibet Plateau and the climatic oscillation of the Quaternary glaciation had a great influence 2of24 | DU et al.