Identification of spider mites based on morphological characteristics is difficult because the morphological differences are subtle and are usually specific to males, which are hard to find, and the adult stage. As an alternative method, we attempted to discriminate the 13 known Tetranychus species in Japan using only females, and esterase and phosphoglucomutase (PGM) zymograms. Using esterase zymograms we were able to discriminate females from five sympatric species (T. ludeni, T. kanzawai, T. piercei, T. pueraricola and T. urticae (red form)) living on kudzu vine, Pueraria lobata, although a sixth sympatric species, T. parakanzawai, was not distinguishable from the closely related T. kanzawai. However, using PGM zymograms, females of each of the 13 species could be identified by the presence of one or two unique alleles. Thus, PGM isozyme analysis is a useful and simple tool for discriminating all Japanese species of the genus Tetranychus.