Historical drainage patterns adjacent to the Qinghai-Tibetan Plateau differed markedly from those of today. We examined the relationship between drainage history and geographic patterns of genetic variation in the Yunnan spiny frog, Nanorana yunnanensis, using approximately 981 base pairs of mitochondrial DNA partial sequences from protein-coding genes ND1 and ND2, and intervening areas including complete tRNA(Ile), tRNA(Gln) and tRNA(Met). Two null hypotheses were tested: (i) that genetic patterns do not correspond to the development of drainage systems and (ii) that populations had been stable and not experienced population expansion, bottlenecking and selection. Genealogical analyses identified three, major, well-supported maternal lineages, each of which had two sublineages. These divergent lineages were completely concordant with six geographical regions. Genetic structure and divergence were strongly congruent with historical rather than contemporary drainage patterns. Most lineages and sublineages were formed via population fragmentation during the rearrangement of paleodrainage basins in the Early Pliocene and Early Pleistocene. Sympatric lineages occurred only in localities at the boundaries of major drainages, likely reflecting secondary contact of previously allopatric populations. Extensive population expansion probably occurred early in the Middle Pleistocene accompanying dramatic climatic oscillations.