Clarifying the genetic structure and population history of a species can reveal the impacts of historical climate and geological changes, providing critical insights for developing effective conservation strategies for ecologically significant fish. The Markakol grayling (), an endangered species found in the Altai-Sayan Mountain region of Central Asia, serves as an ideal model for studying these factors. In this study, populations of a grayling () species discovered in the upper Irtysh River headwaters in Xinjiang, China, were analyzed to assess genetic diversity and population structure. Mitochondrial DNA sequences (cytochrome b and control region), along with 10 microsatellite markers, were used to examine genetic variation. Phylogenetic and genetic distance analyses confirmed the species, long misidentified as Arctic grayling (), as . This species can be divided into two distinct geographic groups: eastern and western, with the Crane River acting as the boundary. The divergence between these groups likely corresponds to refugia formed during the Pleistocene glaciation of the Altai Mountains, approximately 0.48 MA (million years ago) (range: 0.30 to 0.71 Ma). High haplotype diversity (Hd > 0.5) and low nucleotide diversity (π < 0.005) suggest that, despite the species' genetic richness, remains vulnerable to genetic drift, which could threaten its long-term survival. This vulnerability may stem from inbreeding within small refugial populations during the glacial period, followed by gradual population expansion. Our study offers novel insights into grayling populations, with results that have direct implications for management by serving as a tool for the identification of conservation units.