A high degree of genetic diversity among 29 strains of Prevotella (Bacteroides) ruminicola from the rumen was revealed by comparing restriction fragment length polymorphisms in 16S rRNA genes, sodium dodecyl sulfate-polyacrylamide gel profiles of total-cell proteins, and G + C contents of chromosomal DNAs. In order to obtain information on phylogenetic relationships, the sequences of a 389-bp region of the 16S rRNA gene, including variable regions 4 and 5, were compared for 10 strains. These 10 strains formed a single group when their sequences were compared with 16S ribosomal DNA sequences from other species, including Bacteroides spp. from the human colon. On the other hand, the great genetic distances between many P. ruminicola strains, including P. ruminicola subsp. brevis B(1)4 and GA33 and P. ruminicola 23T (T = type strain), support the hypothesis that these organisms should be reclassified into new species. We identified signature oligonucleotides based on 16S ribosomal DNA sequences that distinguished strains related to strains 23T, B(1)4, GA33, and M384, as well as an oligonucleotide that specifically recognized all but one of the Bacteroides and Prevotella strains tested. On the basis of the priming activities of these signature oligonucleotides in PCR reactions and on other criteria, we concluded that 12 of the original 29 strains were related to strain 23T, 4 were related to strain B(1)4, and 4 were related to strain GA33. While there are clear grounds for subdividing the species P. ruminicola on the basis of genotypic differences, it is appropriate to delay formal reclassification until further work on the phenotypic differentiation of the new groups is completed.