Brachymystax lenok (fine-scaled lenok) is a rare and endangered fish species found in the Irtysh River basin. In this study, we utilized mitochondrial Cyt b gene and D-loop (control region) sequences to analyze the population genetic structure of B. lenok in the Irtysh River, providing a theoretical basis for preserving its genetic diversity. Results indicated that 13 haplotypes (Hap1-13) were detected in the combined sequences of Cyt b (1063 bp)+D-loop (1089 bp) across 86 samples from 4 populations. Among these, Hap1 was centrally located in the haplotype network and widely distributed across populations, suggesting it is the ancestral haplotype. The haplotype diversity index (H_d) was 0.515, and the nucleotide diversity index (P_i) was 0.00040, indicating a low level of population genetic diversity. Analysis of molecular variance (AMOVA) revealed that the genetic variation was primarily within populations (99.51%), with variation among populations accounting for only 0.49%, indicating a low level of genetic differentiation (F_st=0.00491, P＞0.05) and no significant genetic differentiation among populations. The mismatch distribution curve showed a unimodal distribution, and the neutral test of Tajima’s D (−2.022) and Fu’s F_s (−9.530) were negative (P＜0.05), and the test of SSD and Hir were not significant (P＞0.05), suggesting that the B. lenok population in the Irtysh River underwent a historical expansion. Bayesian skyline plot (BSP) analysis indicated that the expansion occurred approximately 0.039 million years ago (Ma) and that the Irtysh River populations exhibited significant genetic differentiation from populations in other basins, such as the Heilongjiang (Amur River), Lena River, and Yenisei River, suggesting that the evolutionary history of these populations may be related to historical geological events such as the Sayan-Altaic orogeny, the Quaternary glaciation, and ancient hydrological changes in the Altai Mountains. We concluded that the B. lenok population in the Irtysh River basin can be identified as a distinct evolutionarily significant unit (ESU). It is recommended to effectively protect its natural spawning grounds and breeding populations, restore river connectivity to maintain gene flow between populations, avoid small population breeding during artificial propagation to prevent genetic drift and a reduction in genetic diversity, and prohibit the introduction of non-native stocks from other basins to prevent genetic pollution.