Abstract: Botiidae (Actinopterygii: Cypriniformes) is a group of small and medium-sized fish with eight genera and 62 species, which is widely distributed in Southeast Asia, East Asia and South Asia. A total of 240 sequences were obtained by PCR amplification and downloading from GenBank, covering eight genera and 20 species of Botiidae to clarify the phylogenetic relationships and the origin evolution of the family Botiidae. In this study, phylogenetic tree reconstruction, divergence time estimation and biogeographic analysis were performed using the combination of two mitochondrial genes (CO I, Cytb) and three nuclear genes (IRBP, RAG1, RH). The topology of Maximum Likehood (ML) and Bayesian inference (BI) trees based on different datasets consistently showed that the family Botiidae was a monophyletic taxa and all genera were also monophyletic taxa. The family Botiidae was divided into two major brances of the subfamily Botiinae and Leptobotiinae, of which Botiinae included Sinibotia, Ambastaia, Yasuhikotakia, Syncrossus, Botia and Chromobotia and Leptobotiinae included Parabotia and Leptobotia. The above results were well supported. The results of divergence time and geographic ancestral reconstruction indicated that the family Botiidae originated in Southeast Asia in the Early Oligocene (about 28.85 million years ago), and the genera began to diverge in the Miocene (about 24.25 million years ago), with obvious divergence of species within genera in the Late Miocene and Pliocene (about 17.17-3.79 million years ago). With the movement of the Himalayas, the family Botiidae spread and evolved westward and northward from Southeast Asia, among which the genus Botia entered the South Asian region westward, and the genera of Leptobotia, Parabotia, and Sinibotia entered the Yangtze River and Pearl River basin in China to the north. The differentiation of the family Botiidae was closely related to the movement of the Himalayas. This research has important implications for the phylogenetic relationships and evolutionary processes in the origin of Botiidae.