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A total of 382 stool samples were examined during a survey of intestinal parasites in members of the Tapirapé ethnic group, who live in the Brazilian Amazon region of Mato Grosso. Fecal DNAs from Blastocystis-positive samples were extracted, polymerase chain reaction amplified using Blastocystis-specific primers targeting the small subunit rRNA gene, and sequenced. Three subtypes (STs) were identified: ST1 (41%), ST2 (32%), and ST3 (17%). Seven mixed infections were found (11%). The subtype distribution was markedly different from that reported in Europe in that ST4 was not detected and ST3 was not the most common subtype. This study is the first to include molecular characterization of Blastocystis in Brazil and in indigenous communities from Latin America.