BACKGROUND: Neodon, a typical group of high-altitude rodents, originated in the high-altitude regions of the Tibet-Himalayan area. These rodents inhabit meadows, swampy grasslands, and shrublands at elevations ranging from 3,700 to 4,800 m in Qinghai-Tibet Plateau, where they experience environment stresses such as hypoxia, enhanced ultraviolet radiation, and low temperature. RESULTS: In this study, we assembled a high-quality chromosome-level genome of Neodon fuscus and use it to investigate the potential genetic bases of high-altitude adaptation. The genome of N. fuscus shows gene family expansion in genes related to immunity. Comparative genomics analysis revealed several positively selected genes and rapidly evolving genes associated with DNA damage repair (Atm and Pdia4) and male sperm quality (Idh3b and Atplb1) that aid in clarifying high-altitude adaptation of Neodon species. Additionally, we found that genes (Nrp2, Vav3, Cat and Adam8) associated with oxidative stress and cardiovascular regulation seem to have experienced convergent evolution among plateau species. Notably, we identified a Neodon-specific 24 bp deletion in Map3k6, which may regulate angiogenesis, aiding Neodon species adapt to hypoxic environments. CONCLUSION: These findings elucidate molecular mechanisms underlying high-altitude adaptation, emphasizing the integrative roles of immunity, stress response, and reproduction. The high-quality genome of N. fuscus offers new insights into how Neodon adapt to high-altitude environments and will facilitate future research on Neodon. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-11709-4.