Spider mites are difficult to identify because they are very small and have a limited number of diagnostic characters. Most species of the spider mite genus Tetranychus in Japan are morphologically similar, differing only in the diameter of the aedeagal knob in males. Because this genus contains many important pests, the unambiguous identification of species is crucial for effective pest management and quarantine procedures. DNA-based methods could complement the morphological methods. We examined whether Tetranychus species in Japan could be identified by DNA sequences using the internal transcribed spacer region of nuclear ribosomal DNA and the cytochrome c oxidase subunit I gene of mitochondrial DNA. We determined sequences of the 13 known Tetranychus species in Japan. We could identify 10 of the 13 species in the internal transcribed spacer tree. In the cytochrome c oxidase subunit I tree, we could identify all 13 known Tetranychus species in Japan. Although Tetranychus kanzawai Kishida and T. parakanzawai Ehara were identified by DNA sequences, they were clearly separated into two monophyletic clades each, indicating that a cryptic species existed in each species.