Anaerobic chytrid strains (representing four genera and seven species based on morphological characters), obtained from a variety of hosts, were analysed for variability within the 5' terminal segment of the ribosomal large subunit (LSU) gene, first internal transcribed spacer (ITS1), and the intergenic spacers (IGS) of the ribosomal repeat unit. In the analyses of the ribosomal LSU gene, strains of Neocallimastix, Orpinomyces, and Anaeromyces clustered into distinct groups, but Piromyces strains did not. Although the size of polymerase chain reaction products for the ITS1 region varied among strains, restriction fragment length polymorphism analysis was not useful in distinguishing between the strains. In the analyses of restriction fragment profiles of the IGS, strains of Orpinomyces and Anaeromyces clustered into distinct groupings that corresponded with morphological characters. In contrast, Neocallimastix strains clustered into two distinct groups. Considerable variability was observed in the IGS for strains of Piromyces. Analysis of this region split strains of Piromyces rhinzinflata obtained from a single bison into at least two distinct genetic groupings. Furthermore, the Piromyces strains obtained from bovine, wapiti, and moose clustered according to host. Overall, our molecular data suggest that morphological characters as observed under the light microscope are insufficient for the designation of phylogenetic species of Neocallimastix and Piromyces, and that integration of molecular and morphological data will be required to establish a natural classification system for the anaerobic members of the Chytridiomycota.