Three bovine amphistomes, Calicophoron calicophorum, Orthocoelium streptocoelium and Homalogaster paloniae, are common in Japan. This study was carried out to describe ITS2 sequences in the 3 species, and to evaluate a PCR-RFLP technique based on ITS2 sequence for the species identification in single eggs of the parasites. The ITS2 sequences of the three species contained 19 variable sites including one gap. The sequence difference between species was 4.2-5.3%. The three species of amphistomes were identified based on the difference in the restriction sites of Acc II on the ITS2 sequence.