Speciation remains a fundamental issue in biology. Herein, we report an investigation into speciation in the Rana chensinensis species complex using DNA sequence data from one mitochondrial and five nuclear genes. A phylogenetic analysis of the data revealed four major clades in the complex, and each of them was found to likely represent a species, including one cryptic species. Ecological niche models were generated from 19 climatic variables for three of the four major clades, which were represented by widespread sampling, including R. chensinensis, Rana kukunoris and the potential cryptic species. Each clade is associated with a unique ecological unit, and this indicates that ecological divergence probably drove speciation. Ecological divergence is likely related to the late Cenozoic orogenesis of the Qinghai–Tibetan Plateau. In addition, gene flow between species was detected but only in peripheral portions of the ranges of the four major clades, thus likely had little influence on the speciation processes. Discordances between mitochondrial and nuclear genes were also found; the nominal species, R. chensinensis, contains multiple maternal clades, suggesting potential mitochondrial introgression between R. chensinensis and R. kukunoris.