The Chinese population genetic structure of the twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), was investigated by amplifying and sequencing a portion (453 bp) in the mitochondrial DNA gene coding for cytochrome oxidase I (COI). The study was aimed at checking genetic difference among the Chinese populations of the mite and analyzing its possible dispersal within China. Thirteen populations from east, west, northeast, northwest, and central parts of mainland China were sampled. In the comparisons of these sequences, 36 nucleotide positions (7.9% of the total length) were found polymorphic, of which 88.9% were silent substitutions. Intraspecific variation analysis showed that the Xinjiang (XJ) sample differed from the other samples by an average of 4.68% nucleotide divergence, which was represented two different phylogenetic lineages in the phylogenetic tree. Apart from the XJ sample, the other samples displayed little variation (nucleotide divergences below 1.77%) with each other and clustered in one of the lineages. We argue that the reasons for the remarkable distinction are probably geographical barriers and the use of different host plants.