Background <p>Mongolian gazelle (<i>Procapra gutturosa</i>) populations have experienced significant range contraction and fragmentation over the past century due to habitat loss and barriers. This raises concerns about genetic diversity and connectivity. Understanding the genetic structure of these gazelles across their range is critical for effective conservation management.</p> Result <p>We used mitochondrial DNA sequences and microsatellite markers to assess genetic diversity and population structure in Mongolian gazelles from Mongolia and northern China. Gazelles in Mongolia exhibited high mitochondrial diversity and moderate microsatellite diversity overall. In contrast, gazelles in the Hulun Lake Reserve (HLH) had lower genetic diversity (e.g., reduced mitochondrial haplotype diversity) compared to gazelles on the contiguous Mongolian steppe. Genetic differentiation was low between gazelles along the China-Mongolia border and those in Mongolia. However, the HLH population was moderately differentiated from the Mongolian population and exhibited a distinct genetic structure. These results indicate that the HLH gazelles are relatively isolated and genetically distinct from other Mongolian gazelle populations.</p> Conclusion <p>Fragmentation has led to detectable genetic subdivision in this once panmictic species. To maintain overall genetic health, it is essential to preserve landscape connectivity and gene flow between subpopulations. Conservation measures, such as protecting migratory corridors or introducing individuals from the larger Mongolian population into isolated groups like HLH, could help bolster genetic diversity and reduce inbreeding. Our findings underscore the need for transboundary collaboration to ensure the long-term viability of the Mongolian gazelle.</p>