Scrub typhus is one of the important vector borne illness which is largely underdiagnosed, particularly in children. It causes mild febrile illness to severe complications. More than 20 genotypic clusters are documented from various geographical regions based on sequence variations of 56kDa type specific antigen gene of Orientia tsutsugamushi, the causative agent of scrub typhus. Adequate knowledge about epidemiology and genetic diversity in endemic regions is an important tool for clinical management, development of diagnostic kit and vaccines. Limited studies are available based on genotypic characterization of Orientia tsutsugamushi in children. The present study determined the prevalence and genotypic characterization of Orientia tsutsugamushi in febrile pediatric patients admitted in tertiary care hospital of Chennai, South India. Both serum and blood samples were collected from 239 scrub typhus suspected febrile pediatric patient’s aged between 6 months to 12 years. IgM ELISA and 56kDa nested PCR were performed on all the patient samples. Nested PCR positive samples were sequenced and analyzed for genotypic differences. Among 239 samples, 103 were positive for IgM ELISA and 35 were positive for nPCR analysis. Out of the 108 scrub typhus positive cases, 45.31% (58/128) were male and 45.05% (50/111) were female. Eschar was positive in 56.48% of patients. Pneumonia (4/108), hypotensive shock (3/108), and myocarditis (1/108) were the most common clinical complications associated with scrub typhus positive children. Karp (56.6%) was the most common genotypic cluster found in our study, followed by TA716 (33.33%), TA763 (2/30), and Gilliam (1/30).