The diversity and distribution of freshwater rotifers have been under-explored in China, especially from high altitude regions. We used molecular data to explore these points in the monogonont rotifer Brachionus calyciflorus species complex across China, covering both the low- and high-altitude regions. Populations of this species complex were detected in 44 of the 251 waterbodies sampled across China. Analysis of partial sequences of the nuclear ribosomal internal transcribed spacer (ITS-1) placed these populations in four distinct species ( B. dorcas , B. elevatus , B. calyciflorus sensu stricto ( s.s. ) and B. fernandoi ), and phylogenies based on sequences from a portion of the mitochondrial cytochrome c oxidase subunit I gene recognized seven groups within the B. calyciflorus species complex in China. The four species were congruent with the four morphogroups that we identified. All four species were present in the Eastern Plain and B. dorcas occurred nowhere else. Brachionus elevatus and B. calyciflorus s.s. were the only species present in the Northeast Plain and in the Yunnan-Guizhou Plateau, respectively. Cases of mito-nuclear discordance were detected in our specimens, suggesting occasional hybridization between different species of the B. calyciflorus species complex. Our results revealed the phylogeography and gene introgression in this species complex across China.