Three spider mite species, Eotetranychus sexmaculatus, Eutetranychus orientalis and Oligonychus biharensis, are widely distributed in China, infesting rubber trees. They cause various damages in different areas with different landforms, climates and rubber-growing varieties. The difference in habitat of the spider mites may result in genetic differentiation. It supposed be one of the main reasons for their various damage levels in different geographic regions. To examine whether the population genetic differentiation exist between E. sexmaculatus, E. orientalis and O. biharensis, we collected samples of three mite species from ten locations in China and one in Vietnam. Mitochondrial COI sequences of collected samples were analyzed. The results showed that the value of genetic differentiation among populations of the three mites was: E. orientalis > O. biharensis > E. sexmaculatus. There were 16 haplotypes in eight populations of E. orientalis and relatively large genetic distances between haplotypes. E. orientalis had the maximum Fst and Gst values, 0.792 and 0.434, respectively. There was no significant differentiation of E. sexmaculatus among different geographic regions after excluding the Ruili population. Both O. biharensis and E. orientalis were clustered into three large branches in the phylogenetic tree. A single peak appeared in the total population mismatch distribution curve and a star-like haplotype network of E. sexmaculatus suggests a history of population expansion. Three rubber tree spider mites collected in Yunnan had the greatest genetic diversity with the maximum number of haplotypes. In contrast, the genetic diversity of these three spider mites collected in Hainan and Guangdong was relatively small, with few haplotypes. These results implied that genetic differentiation existed in these three spider mites. Specific property and geographic isolation might be the major factor triggering genetic differentiation of E. orientalis, E. sexmaculatus and O. biharensis.