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Geographical barriers and distance can reduce gene exchange among animals, resulting in genetic divergence of geographically isolated populations. The Tibetan antelope (Pantholops hodgsonii) has a geographical range of approximately 1600 km across the Qinghai-Tibet Plateau, which comprises a series of tall mountains and big rivers. However, previous studies indicate that there is little genetic differentiation among their geographically delineated populations. To better understand the genetic structure of P. hodgsonii populations, we collected 145 samples from the 3 major calving regions, taking into consideration their various calving grounds and migration routes. We used a combination of mitochondrial sequences (Cyt b, ATPase, D-loop and COX I) to investigate the genetic structure and the evolutionary divergence of the populations. Significant, albeit weak, genetic differentiation was detected among the 3 geographical populations. Analysis of the genetic divergence process revealed that the animals gradually entered a period of rapid genetic differentiation approximately 60 000 years ago. The calving migration of P. hodgsonii cannot be the main cause of their weak genetic structure because this cannot fully homogenize the genetic pool. Instead, the geological and climatic events as well as the coupling vegetation succession process during this period have been suggested to greatly contribute to the genetic structure and the expansion of genetic diversity.