The present study describes the molecular epidemiology of Human herpesvirus 8 (HHV-8) among four Indian tribes (Kararao, Arara Laranjal, Tiriyo, and Zo'e) of the Amazon region of Brazil and a group of HIV-1-infected subjects from the urban population of Belem, Para. Infection was characterized by the presence of antibodies using ELISA (measuring antibodies to ORF59, ORF65, K8.1A, K8.1B, and ORF73), and molecular assays (gene amplification of the regions ORF26 and the variable region VR1). Antibodies to HHV-8 were detected in 66 samples of the 221 Brazilian Amerindians, namely, 6 (25%) in the Kararao, 18 (19.6%) in the Arara Laranjal, 24 (42.9%) in the Tiriyo, and 18 (36.7%) in the Zo'e. Among the 477 HIV-1-infected subjects, antibodies to HHV-8 were present in 74 (15.5%) persons. The ORF26 region was amplified in seven samples, one of the Arara Laranjal, one of the Tiriyo, two of the Zo'e, and three of the HIV-1-infected group. Subtyping of HHV-8 described a high multiplicity of molecular subtypes, including C (Zo'e), E (Tiriyo), and B (HIV-1 infected). Serological results confirm the high prevalence of HHV-8 among Amerindians and the presence of three subtypes in the Amazon region of Brazil, including a unique subtype, which favors the idea of HHV-8 as an ancient human infection within this particular geographical region.