Rickettsioses caused by Rickettsia and Orientia spp. are the re-emerging diseases in India, which are grossly underdiagnosed, particularly among children. They usually present as mild-febrile illness but may extend to severe life-threatening complications. Early diagnosis followed by proper treatment reduces the morbidity and mortality. Non-specific clinical symptoms and lack of point of care diagnosis may delay the treatment. Molecular assays like PCR may helpful in the early diagnosis and confirmation of rickettsial diseases. In this study, we used multiplex real-time PCR to detect Rickettsia spp. and Orientia spp. in febrile pediatric patients. Whole blood was collected from 239 clinically suspected febrile pediatric patients aged between 6 months to 12 years admitted in tertiary care hospital at Chennai, South India. Multiplex real-time PCR was used to target the gltA gene for Rickettsia spp. and the 47kDa gene for Orientia tsutsugamushi. To compare the sensitivity, nested PCR was performed on the 56kDa antigen gene of O. tsutsugamushi and the Rickettsia genus specific gltA gene. By multiplex real-time PCR, 15 samples were positive for O. tsutsugamushi and 3 were positive for Rickettsia spp. Nested PCR identified 35 positive samples for O. tsutsugamushi and 4 positive samples for Rickettsia spp. Even though multiplex real-time PCR had lower positivity than nPCR, it was effective in diagnosing O. tsutsugamushi and Rickettsia spp. in a single assay.