The genus Oligonychus has been morphologically divided into two groups based on the direction of curvature of the aedeagus and includes some morphologically similar species that are difficult to distinguish. To develop DNA-based methods for identifying Oligonychus species and to determine the phylogenetic relationships among them, we examined the cytochrome c oxidase subunit I gene of mitochondrial DNA and the internal transcribed spacer and 28S regions of nuclear ribosomal RNA gene for 17 species. Based on the genetic distances (p-distances) of the three DNA regions, the range of intraspecific divergence was found to be below (and not overlap) the range of interspecific divergence, which allowed the 17 species to be discriminated correctly, consistent with their classification based on morphology. Phylogenetic trees constructed by neighbor-joining and Bayesian methods clearly showed two clades, consisting of species whose aedeagi curve ventrally and dorsally, respectively. Three Oligonychus species inhabiting gramineous plants formed clearly defined subclades.