The phylogenetic relationships of the microsporidian species Microgemma caulleryi, Pleistophora finisterrensis and Tetramicra brevifilum were investigated on the basis of restriction fragment length polymorphism (RFLP) analysis of PCR-amplified small-subunit rDNA (SSUrDNA). Using PCR primers specific for microsporidian SSUrDNA, a single product was obtained from each species, and heteroduplex analysis indicated a high degree of sequence homology among the 3 products. In RFLP analysis of the PCR-amplified SSUrDNA, the enzymes AluI and DdeI gave restriction patterns that differed among all 3 species. Phylogenetic analysis using restriction patterns as differential characters indicated that Microgemma caulleryi and Tetramicra brevifilum are more closely related to each other than to Pleistophora finisterrensis.