We attempted a population survey of spider mites and phytoseiid mites in Japanese peach orchards with different pesticide practices; however, we had difficulty discriminating phytoseiid mites. To estimate phytoseiid mite species composition, ribosomal gene fragments were amplified from genomic DNA of five phytoseiid mite species using PCR. Cloning and nucleotide sequencing of amplified fragments identified species-specific polymorphic sites. Newly amplified fragments from recombinant plasmids were mixed in various ratios to produce standard DNA template mixtures. After direct sequencing, the signal ratios between two nucleotides at each species-specific polymorphic site were calculated and shown against the corresponding expected ratios. Quadratic regression equations were used to estimate the phytoseiid mite species composition. Results showed that the phytoseiid mite species composition changed during the survey period and varied among study sites.