Abstract: In order to understand the genetic resources of Mastacembelus armatus populations in southern China, and to provide an important scientific basis for their management and protection, we collected 140 individuals from 16 geographical populations from seven independent river systems in southern China and sequenced two mitochondrial genes (COI and Cytb) via PCR amplification and Sanger sequencing, and finally revealed the genetic structure and demographic history of M. armatus populations by phylogenetic analysis, haplotype network, population genetic analysis and Bayesian skyline plot. The results show that M. armatus populations consisted of three lineages (I, II and III) and split between 0.596 and 0.676 million years ago (Ma). Haplotype network shows that there was a common domain distribution among different lineages, and suggests that there might be two diffusion routes between Hainan Island population and mainland population. Population genetic analysis finds significant genetic differentiation (F_ST=0.676, P<0.001) and isolation by distance pattern (R=0.463, P=0.001) among M. armatus populations, implying that spatial distance was an important factor for genetic differentiation of M. armatus. Demographic analysis shows that M. armatus populations experienced population expansion at 0.025 Ma.