Scrub typhus (ST) is an underdiagnosed acute febrile illness in the Asia Pacific region with recent reemergence. Clinical diagnosis is difficult, and laboratory confirmation is largely based on serological and molecular tests. However, Weil–Felix test still remains the only test available in much of the rural tropics where a disproportionate number of cases occur. Sensitive and affordable assays are important for broader use and accurate diagnosis. We evaluated the diagnostic capabilities of serological and molecular assays on single acute clinical samples. Out of 1036 cases, 319 were confirmed as ST, and the sensitivities of immunofluorescent assay (IFA), IgM enzyme-linked immunosorbent assay (ELISA), nested polymerase chain reaction (n-PCR) and WFT were 93.4%, 80.3%, 75.2%, and 54.2%, respectively. IgM ELISA + n-PCR combination demonstrated highest degree of agreement (κ = .911) in the absence of IFA. Additionally, 16 cases were detected by n-PCR only. Our study emphasizes the diagnostic challenges in the developing world, importance of molecular tests, and best alternate assays in ST diagnosis.