The seroprevalence of human herpesvirus 8 (HHV-8) infection in the Thai population was investigated. Sera from 1,018 human immunodeficiency virus 1 (HIV-1)-negative and 436 HIV-1-positive individuals were tested for antibodies to latent and lytic HHV-8 antigens by indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA) using mixed recombinant orf HHV-8 proteins. The positive sera were further tested with recombinant HHV-8 protein expressed 293T cells by IFA. The seroprevalence of HHV-8 infection was determined by the concordant reactivity of sera among antibody testing assays. The results showed a low rate of HHV-8 seropositivity in both HIV-1-negative healthy individuals (0.6%) and HIV-1-infected patients (0.7%). These results are consistent with the fact that a small number of patients with AIDS-associated KS have been reported in Thailand and that HHV-8 is an uncommon pathogen in this country. Interestingly, we found that sera from the general population living in the north, but not other regions of Thailand, had antibodies to HHV-8.