For diagnosis of melioidosis, we compared polymerase chain reaction (PCR)-based DNA detection and three serologic methods with the culture method currently used as gold standard. The diagnostic values of the serologic methods were evaluated in 130 patients. All these patients resided in an endemic area. An enzyme-linked immunosorbent assay (ELISA) gave slightly higher specificity (86.2%) than a dot immunoassay (DOT) (85.3%), but was superior to an indirect hemagglutination assay (IHA) (79.8%). The sensitivities of the DOT (85.7%) and ELISA (71.4%) were considerably higher than that of IHA (61.9%). However, the PCR was the most sensitive (95.2%) and specific (91.7%). Nevertheless, DOT and ELISA are more practical for local hospitals. With the high negative predictive value of both the ELISA (94.0%) and DOT (96.9%) in a high prevalence area, clearly these methods can rule out most of the non-melioidosis patients.