Toxoplasma gondii infection was studied in Khon Kaen, northeastern Thailand. Blood specimens were collected from 493 non-pregnant women between 2009 and 2012. Presence of antibodies to T. gondii was determined using the latex agglutination test (cutoff titer 1:32). Thirteen of 493 (2.6%) were found to be seropositive for T. gondii infection. There was no age dependence in the prevalence. The present study reporting a low prevalence of T. gondii infection in northeastern Thailand suggests that the traditional lifestyles and climatic environments may limit the emergence of toxoplasmosis in this region.