Sequences of small-subunit rRNA genes have been obtained for four new isolates of Entamoeba. Phylogenetic analyses give new insights into the evolution of these organisms. A novel Entamoeba from pigs in Vietnam that produces uninucleate cysts proved to be unrelated to other uninucleated cyst-producing species. Revival of the name Entamoeba suis for this organism is proposed. Instead of being related to Entamoeba polecki, it shares a recent common ancestor with the non-encysting Entamoeba gingivalis in a lineage that is basal to the tetranucleate cyst-producing clade. This suggests that species producing cysts with four nuclei are descended from an ancestor that produced cysts with a single nucleus. An Entamoeba from a horse was isolated in culture. No cysts were observed in the original stool sample but the sequence is placed unequivocally within the clade of tetranucleate cyst-producing species with no other sequences being specifically related. Revival of the name Entamoeba equi for this organism is proposed. The Entamoeba ecuadoriensis sequence was found to be the most closely related to Entamoeba histolytica and Entamoeba dispar, as predicted, despite the organism having been an environmental isolate originally assigned to Entamoeba moshkovskii. Finally, a partial E. polecki gene sequence from a pig proved to be virtually identical to that of Entamoeba struthionis from an ostrich, suggesting that the latter name is a synonym.