Background: Entamoeba species harbored by humans have diferent degrees of pathogenicity. The present study explores the intra- and interspecifc diversity, phylogenetic relationships, prevalence and distribution of tetra- and octonucleated cyst-producing Entamoeba in diferent Brazilian regions. Methods: Cross-sectional studies were performed to collect fecal samples (n=1728) and sociodemographic data in communities located in four Brazilian biomes: Atlantic Forest, Caatinga, Cerrado, and Amazon. Fecal samples were subjected to molecular analysis by partial small subunit ribosomal DNA sequencing (SSU rDNA) and phylogenetic analysis. Results: Light microscopy analysis revealed that tetranucleated cysts were found in all the studied biomes. The highest positivity rates were observed in the age group 6–10 years (23.21%). For octonucleated cysts, positivity rates ranged from 1 to 55.1%. Sixty SSU rDNA Entamoeba sequences were obtained, and four diferent species were identifed: the octonucleated E. coli, and the tetranucleated E. histolytica, E. dispar, and E. hartmanni. Novel haplotypes (n=32) were characterized; however, new ribosomal lineages were not identifed. The Entamoeba coli ST1 subtype predominated in Atlantic Forest and Caatinga, and the ST2 subtype was predominant in the Amazon biome. E. histol ytica was detected only in the Amazon biome. In phylogenetic trees, sequences were grouped in two groups, the frst containing uni- and tetranucleated and the second containing uni- and octonucleated cyst-producing Entamoeba species. Molecular diversity indexes revealed a high interspecifc diversity for tetra- and octonucleated Entamoeba spp. (H±SD=0.9625±0.0126). The intraspecifc diversity varied according to species or subtype: E. dispar and E. histo lytica showed lower diversity than E. coli subtypes ST1 and ST2 and E. hartmanni. Conclusions: Tetra- and octonucleated cyst-producing Entamoeba are endemic in the studied communities; E. histolytica was found in a low proportion and only in the Amazon biome. With regard to E. coli, subtype ST2 was predominant in the Amazon biome. The molecular epidemiology of Entamoeba spp. is a feld to be further explored and provides information with important implications for public health.