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Differentiation of Blattella asahinai Mizukubo, Blattella vaga Hebard, and Blattella germanica (L.) was investigated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). DNA fragments corresponding to mitochondrial CO1,CO1/TL2(small and large fragments), and ITS2 regions were amplified using PCR. The universal primers used for amplifying mitochondrial CO1 and CO1/TL2 (small and large fragments) were CO1-J-1718/CO1- N-2191, CO1-J-2441/TL2-N-3014, and CO1-J-1751/TL2-N-3014. Amplification was observed with all primer combinations, but diagnostic patterns were found only with AluI digested amplicons of CO1 and CO1/TL2 (large fragments) regions. Both inter/intraspecific polymorphism was observed for the 517 bp CO1 region between German (A, B) and Asian (A, B, C) cockroach populations. Polymorphic form B of the Asian cockroach (374 and 179 bp fragments) was nearly identical to the field cockroach (370 and 178 bp fragments). The CO1 amplicon (517 bp) differentiated German and field cockroaches but not Asian and field cockroaches. The CO1/TL2 amplicons (1275 bp) digested with AluI clearly differentiated all three species. The Asian cockroach had a distinct 540 bp fragment, whereas the field cockroach had a 403 bp fragment and the German cockroach lacked both fragments. Intraspecific polymorphs for CO1/TL2 amplicons (1275 bp) were observed with the German cockroach only. Portions of the CO1 and TL2 genes were sequenced, revealing 76% identity among German, Asian, and field cockroaches. Sequence data also revealed that German and Asian cockroaches were more closely related to each other than to the field cockroach.