Despite the species' wide distribution, studies of the genetic diversity within Entamoeba coli and Entamoeba hartmanni remain limited. In the present study, we provide further insight into the genetic diversity of both species based on analysis of partial nuclear small subunit ribosomal DNA sequences generated from human fecal DNAs from samples collected in Africa, South America, and Europe. Reinforcing the previous recognition that E. coli is a species complex, our data confirm the existence of the two subtypes, ST1 and ST2, previously identified plus, potentially, a new subtype, ST3. While ST1 appears to be genetically quite homogenous, ST2 shows a substantial degree of intrasubtype diversity. ST2 was more common in samples collected outside Europe, whereas ST1 showed no geographical restriction. The potentially novel subtype is represented to date exclusively by sequences from South American and African samples. In contrast to previous reports, our new data also indicate substantial variation in E. hartmanni that could also support the establishment of subtypes within this species. Here, however, no links were identified between subtype and geographical origin.