Genetic differences among six didymozoid species distinguished on morphological criteria (Didymozoon species A, B, E, F and G, and Neometadidymozoon helicis) were sought by restriction fragment length analysis of internal transcribed spacer (ITS) and 5.8S ribosomal DNA. DNA fragments comprising ITS1, 5.8S and ITS2 were amplified by polymerase chain reaction for each of the six species. Digestion with the restriction enzyme (RE) Rsa I yielded four to six fragments, approximately 65-775 base pairs long. Digestion with another RE, Sau 3AI, yielded five to eight fragments, approximately 25-775 base pairs long. No intraspecific variation was detected but there were many interspecific differences. All six species distinguished by morphology are genetically distinct. Thus, the reliability of the morphological characters originally used to separate the species was confirmed.