The tufted deer (Elaphodus cephalophus), a Near-Threatened (NT) species endemic to China and Myanmar, requires robust genetic data for effective conservation. However, the genetic landscape of key populations, such as those in Chongqing, remains poorly understood. This study aimed to comprehensively evaluate the genetic diversity, population structure, gene flow, and demographic history of tufted deer across this critical region. We analyzed mitochondrial DNA (mtDNA) from 46 non-invasively collected fecal samples from three distinct populations: Jinfo Mountain (JF, n = 13), Simian Mountain (SM, n = 21), and the Northeastern Mountainous region (NEM, n = 12). Genetic variation was assessed using the cytochrome b (Cyt b) and D-loop regions, with analyses including F_st, gene flow (N_m), neutrality tests, and Bayesian Skyline Plots (BSP). Our results revealed the highest genetic diversity in the SM population, establishing it as a genetic hub. In contrast, the JF population exhibited the lowest diversity and significant genetic differentiation (>0.23) from the SM and NEM populations, indicating profound isolation. Gene flow was substantial between SM and NEM but severely restricted for the JF population. Demographic analyses, including BSP, indicated a long history of demographic stability followed by a significant expansion beginning in the Middle to Late Pleistocene. We conclude that the SM/NEM metapopulation serves as the genetic core for the species in this region, while the highly isolated JF population constitutes a distinct and vulnerable Management Unit (MU). This historical demographic expansion is likely linked to climatic and environmental changes during the Pleistocene, rather than recent anthropogenic factors. These findings underscore the urgent need for a dual conservation strategy: targeted management for the isolated JF population and the establishment of ecological corridors to connect the Jinfo Mountain and Simian Mountain populations, ensuring the long-term persistence of this unique species.

Effective conservation and management of the endangered tufted deer (Elaphodus cephalophus) necessitate genetic diversity research. This study systematically assessed the genetic diversity, population structure, gene flow, and demographic history of three representative tufted deer populations in Chongqing, China (Jinfo Mountain, Simian Mountain, Northeastern Mountainous area) using combined mitochondrial Cytochrome b (Cyt b) gene and D-loop region analysis. Our findings reveal the D-loop region demonstrates significantly higher genetic variability and a faster mutation rate than Cyt b. The Simian Mountain population consistently displayed the highest haplotype diversity, suggesting its role as a genetic diversity hotspot and crucial hub for regional gene flow. Haplotype network analysis showed frequent gene exchange between Simian Mountain and Northeastern Mountainous populations, while the Jinfo Mountain population exhibits significant genetic isolation, likely due to unique topographical features. Historical demographic analyses suggest a likely slow expansion, with evidence for recent population expansion despite concurrent regional genetic isolation. This study underscores the critical importance of multi-scale genetic markers for deciphering species’ evolutionary histories and provides essential groundwork for tufted deer conservation. We recommend prioritizing Simian Mountain conservation, enhancing Jinfo Mountain genetic management, and maintaining regional population connectivity through ecological corridors and continuous monitoring.