A qualitative syber green real-time PCR with primers designed for a truncated portion of the 56kDa major outer membrane antigen gene of Orientia tsutsugamushi was used to diagnose scrub typhus from the blood or serum of suspected patients. Sixty-six blood and/or sera samples from fever cases, either with high index of suspicion for scrub typhus and/or positive by Weil–Felix test (≥1:160), were tested with the PCR. Specificity of the PCR was confirmed by end point melt curve analysis and sequencing of the amplicons. A nested PCR for determination of the serotypes of O. tsutsugamushi was performed on to the samples. In real-time PCR strong positive fluorescence was obtained in 73% of the suspected samples. Serotype-specific PCR amplification of some of the positive samples was indicative of the Kuroki type whereas the rest were non-responsive to this test. Sequence analyses of PCR amplicons indicated the presence of new, previously undescribed type of O. tsutsugamushi in this region. This one-step real-time PCR can be used for the detection and confirmation of scrub typhus, when used independently or in conjunction with, the Weil–Felix test, which is still the only available detection test for scrub typhus in most parts of the developing world. Elaborate studies need to be taken up to further evaluate its suitability as specific molecular tool for the diagnosis of scrub typhus and to delineate the prevalent strain types in these regions for a clear epidemiological understanding of this emerging infectious disease.