Small subunit ribosomal DNA sequences were obtained by polymerase chain reaction from four trichomonad species: a frog endosymbiont Trichomitus batrachorum, an intestinal endosymbiont of a squamate reptile, Hypotrichomonas acosta and two free-living isolates, Monotrichomonas carabina and Monotrichomonas sp. Molecular trees inferred by distance, parsimony and likelihood techniques identify three well-resolved clusters within the trichomonads, however bootstrap values do not strongly support a particular branching order for these lineages. The first cluster includes the Devescovinidae and the Calonymphidae. The second clade unites Trichomitus batrachorum and Hypotrichomonas acosta. The third cluster embraces all known free-living genera, including Monotrichomonas, and various members of the Trichomonadinae subfamily such as Trichomonas vaginalis, and Pentatrichomonoides scroa. Neither Monocercomonadidae nor the Trichomonadidae as envisaged are monophyletic. Most of the monocercomonads, which possess a rudimentary cytoskeleton, were likely descendants of more complex forms. The study also suggests that the genus Trichomitus is currently polyphyletic, partly explaining the discordant positions of this genus in previous molecular analyses.