The nucleotide sequences of the first internal transcribed spacer (ITS-1), 5.8S gene and second internal transcribed spacer (ITS-2) of ribosomal DNA have been determined for Cylicocyclus nassatus, C. ashworthi and C. insignis. Pairwise comparisons revealed sequence differences between the taxa ranging from 3.8 to 6.2% for the ITS-2 and 2.2-2.7% for the ITS-1. For the ITS-1, the level of the sequence difference between C. ashworthi and C. nassatus (2.2%) was equivalent to that between C. nassatus and C. insignis (2.2%), indicating that C. ashworthi and C. nassatus represent separate species. Theoretical restriction maps were constructed from the sequence data, and a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-linked RFLP) technique was established to unequivocally distinguish C. ashworthi from C. nassatus.