Histomonas meleagridis is the etiological agent of histomonosis or blackhead disease. Recently, genotyping, based on polymerase chain reaction and sequencing of internal transcribed spacer-1 sequences was applied to various isolates originating from fowl. Three genotypes were described: types I and II isolates were associated with clinical disease and probably derived from H. meleagridis, whereas, type III isolates were not disease-associated and likely corresponded to Parahistomonas wenrichi according to morphological observations. However, this latter species has never been characterized at the molecular level and its phylogenetic relationships with other parabasalids remained hypothetical. To confirm the identification of these isolates, small subunit rRNA gene sequences were obtained from representatives of types I, II, and III and analyzed in a broad phylogeny including 64 other parabasalid sequences. From our phylogenetic trees, we confirmed that types I and II isolates were closely related, if not identical, to H. meleagridis, while type III isolates represented P. wenrichi. Both species clustered together with high support. This grouping suggested that speciation leading to these two species inhabiting the same hosts and ecological niche occurred recently in birds. In addition, speciation was likely followed by loss of pathogenicity in P. wenrichi.