A PCR-RFLP-based method of species identification was considered for 5 Japanese species of Orius Wolff flower bugs, Orius strigicollis (Poppius), O. minutus (Linnaeus), O. sauteri (Poppius), O. nagaii Yasunaga, and O. tantillus (Motschulsky). Nucleotide sequences of the internal transcribed spacer 1 of the nuclear ribosomal gene and a portion of mitochondrial cytochrome oxidase subunit I gene were compared among species and recognition sites of diagnostically useful restriction enzymes were examined. The PCR-RFLP analysis using 108 individuals of 26 laboratory strains confirmed that the 5 species could be correctly identified by banding patterns generated using these DNA regions. Because our PCR primers can amplify DNA fragments from DNA template extracted from both freshly killed insects and dried specimens stored at room conditions, the PCR-RFLP-based method was considered useful for analyses of field populations in which researchers store and accumulate field-collected samples before they perform laboratory examinations.