Frequently, Pleistocene climatic cycling has been found to be the diver of genetic structuring in populations, even in areas that did not have continental ice sheets, such as on the Qinghai-Tibetan Plateau (QTP). Typically, species distributed on the plateau have been hypothesized to re-treat to south-eastern refugia, especially during the Last Glacial Maximum (LGM). We evaluated sequence variation in the mitochondrial DNA gene Cytb and the nuclear DNA gene RAG-1 in Rana kukunoris, a species endemic to the QTP. Two major lineages, N and S, were identified, and lineage N was further subdivided into N1 and N2. The geographical distribution and genealogical divergences supported the hypothesis of multiple refugia. However, major lineages and sublineages diverged prior to the LGM. Demographical expansion was detected only in lineage S and sublineage N2. Sublineage N1 might have survived several glacial cycles in situ and did not expand after the LGM because of the absence of suitable habitat; it survived in river islands. Genetic analysis and environment modelling suggested that the north-eastern edge of QTP contained a major refugium for R. kukunoris. From here, lineage S dispersed southwards after the LGM. Two microrefugia in northern Qilian Mountains greatly contributed to current level of intraspecific genetic diversity. These results were found to have important implications for the habitat conservation in Northwest China.