Very few studies have focused on the biogeography and genetic diversity of aquatic invertebrate zooplankton from the Qinghai‐Tibetan Plateau, an important biogeographic hotspot. The geological uplift of this region created special environments requiring adaptive genetic diversification of the resident lineages. Here, we developed and used the first set of high‐resolution microsatellite markers for the Daphnia similis species complex. We screened this species in 23 (out of 303) lakes across China, covering a large geographical and ecological range of habitats. High population clonal diversity of the D. similis species complex was found, regardless of their geographical origin. However, four Tibetan Daphnia sinensis populations showed substantially lower clonal diversity. Interestingly, D. sinensis populations from two regions, Eastern China and the Qinghai‐Tibetan Plateau of Western China, were found to be clearly differentiated from each other. Lakes inhabited by Western D. sinensis were of a lower trophic level, higher altitude, and more likely to freeze in the winter. This study contributes to an understanding of adaptation to their special environments by the freshwater invertebrates in the Qinghai‐Tibetan Plateau.