Factors that determine the genetic structure of species in southwestern China remain largely unknown. In this study, phylogeography and demography of Tylototriton shanjing was investigated from a mitochondrial perspective to address the role of the Quaternary ice ages in shaping phylogeographic history and genetic diversity of Yunnan. A total of 146 individuals from 19 populations across the entire range of the species were collected. We detected four maternal phylogenetic lineages corresponding to four population groups, and found that major glaciation events during the Pleistocene have triggered the intra-specific divergence. Coalescent simulations indicated that the populations retreated to different refugia located in southern Yunnan, northwestern Yunnan, the border region of western Yunnan with Myanmar, and middle-western Yunnan, respectively, during previous glacial periods in the Pleistocene, and these four refugia were not retained during the Last Glacial Maximum. Population expansions occurred during the last inter-glaciation, during which ice core and pollen data indicated that the temperature and precipitation gradually increased, and declines of population sizes started after the beginning of the Last Glacial Maximum when the climate became cooler and dryer. The paleo-drainage system had no contribution to the current genetic structure and the rivers were not dispersal barriers for this salamander.