ABSTRACT: Yao-Feng Tsao, Wen-Wen Lin, Chia-Hao Chang, Takayoshi Ueda, Nian-Hong Jang-Liaw, Ya-Hui Zhao, and Hsiao-Wei Kao (2016) Rose bitterling, Rhodeus ocellatus, is a small cyprinid fish distributed in East Asia. To infer its phylogeography and genetic structure, specimens from Taiwan, China, and Japan were collected, and complete mitochondrial cytochrome b (cyt b) DNA sequences were amplified and sequenced. Phylogenetic analyses identified seven mitochondrial lineages (A-G). Among them, three lineages (A, B, and C) distributed in mainland China. Lineages D, E, and F distributed in Japan, Korea, and Taiwan, respectively. Lineage G distributed in both China and Japan. The results of the Bayesian Binary MCMC analysis (BBM) suggested that the most recent common ancestor of R. ocellatus was from Lower Yangtze region. Divergence times among lineages inferred by molecular clock ranged from 7.55 to 1.44 million years ago. We propose that topography and climate changes by uplift of the Tibetan Plateau in the Late Miocene-Pliocene and the glacial-interglacial cycles in the Pleistocene might account for population expansion and genetic differentiation. Divergence times among lineages A, B, and C in Yangtze River basin ranged from 7.55 to 2.27 million years ago that might result from changes of flow directions of rivers from westward to eastward driven by the uplift of the Tibetan Plateau. The glacial-interglacial cycles in the Pleistocene might further cause population expansion to the northward of lineage G at about 0.19 million years ago. Lineage D in Japan was dispersed from the mainland China before the opening of the Sea of Japan, and lineage F in Taiwan was dispersed from the mainland China through the land bridge in the Pleistocene. Because of the genetic differentiation is statistically significant among populations, protection of genetic diversity and distinctness of R. ocellatus should be considered in the future conservation management.