The human T-lymphotropic virus (HTLV) and associated diseases, adult T cell leukemia and spastic paraparesis, appear to be endemic in southwestern Japan and the Caribbean. This cross-sectional population-based study was conducted to describe the seroepidemiology of HTLV in the Republic of Panama. HTLV antibody was measured by first generation and commercial ELISA tests and confirmed by competitive binding ELISA, a radioimmunoassay for anti-p 24, and Western blot. Of 3,231 subjects ≥15 years of age, 135 (4.2%) had antibody detected in ELISA screening tests, but because only 20% were confirmed positive, HTLV seroprevalence varied from 0.2–2% throughout the Republic. Infection with HTLV clustered in Guaymi Indians living in Bocas del Toro province (9.9% prevalence rate). With the exception of Guaymi Indians, no major geographic, urban/rural, male/female or racial differences in antibody prevalence were observed; specifically, HTLV infection rates were not elevated in black Panamanians. Clustering of infection in an isolated Amerind population must be further investigated. The small proportion of screen-positive sera which confirmed positive illustrates the importance of strict uniform criteria for seropositivity.