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Human T-lymphotropic viruses 1 and 2 (HTLV-1/2) have a worldwide distribution. HTLV-1 has been associated with several diseases, including an aggressive malignant disease known as adult T-cell leukemia/lymphoma and a chronic inflammatory neurological disease called HTLV-1-associated myelopathy, while HTLV-2 has not been definitively associated with diseases. HTLV-2 is most prevalent in specific groups such as injecting drug users and the indigenous population. In Brazil, most studies about HTLV in indigenous are carried out in indigenous communities from the north of the country. Mato Grosso do Sul (MS), Central Brazil, has the second-largest indigenous population in Brazil. However, there is no available data about HTLV infection in this group. We conducted the first investigation of HTLV-1/2 infection prevalence in the indigenous population from Jaguapiru and Bororó villages in Dourados City, MS, to provide the prevalence and molecular characterization of HTLV. For that, a total of 1875 indigenous participated in the study. All the serum samples were screened by an enzyme-linked immunosorbent assay commercial kit for the presence of anti-HTLV-1/2 antibodies. Positive samples were confirmed by HTLV-1/2 Western Blot assay. The HTLV-1 5'LTR region was detected by nested PCR amplification and sequenced by Sanger. Most of the study population declared belonging to Guarani-Kaiowá ethnicity (69.18%), 872 (46.51%), and 1003 (53.49%) were from Jaguapiru and Bororó villages, respectively. The median age of participants was 31 years, and 74.24% were females. Two individuals were detected with HTLV-1 (0.1%; CI 95% 0.1–0.2). The phylogenetic analysis revealed that isolates belong to the Cosmopolitan subtype and the Transcontinental subgroup (HTLV-1aA). The low HTLV-1 prevalence found in this study is similar to that observed among blood donors, and pregnant populations from Mato Grosso do Sul. The absence of HTLV-2 infection among these Brazilian indigenous communities would suggest a distinct behavior pattern from other indigenous populations in Brazil. [ABSTRACT FROM AUTHOR]