Scrub typhus is common among patients with acute fever in rural areas of Thailand. The authors prospectively recruited patients with acute fever from provincial Thai army hospitals. Dot-ELISA test for scrub typhus was done in hospitals and then compared with standard immunofluorescent assay for diagnosis of scrub typhus. Among 178 patients, scrub typhus was diagnosed by immunofluorescent assay in 10 patients (5.61%). The incidence was high in the northeastern and northern regions. Dot-ELISA gave positive results in 4 of 115 patients, while immunofluorescent assay gave positive results in 6 patients (sensitivity = 66.7%). No false positive results of Dot-ELISA were found among 109 patients (specificity = 100%). All patients gave negative results for murine typhus and Thai tick typhus using immunofluorescent assay. Regarding this present study, Dot-ELISA for scrub typhus has a good sensitivity and specificity and can be used in rural hospitals. This test could be useful for diagnosis of scrub typhus in hospitals where immunofluorescent assay is not available.