Introduction This study aims to evaluate the diagnostic performance of polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) methods, and gene sequencing in detecting , the causative agent of scrub typhus, in pediatric patients with acute encephalitis syndrome (AES). Methods The study was conducted at a tertiary care teaching hospital in northern India. In all, 264 children presenting with AES were enrolled between August 2018 and November 2019. Cerebrospinal fluid (CSF), serum, and peripheral blood mononuclear cell (PBMC) samples were analyzed using real-time PCR and ELISA to detect the presence of scrub typhus. The 56 kDa gene sequencing was performed in 15 samples. Results The ELISA for scrub typhus IgM demonstrated the highest cumulative positivity rate at 26.52%. In contrast, PCR testing of CSF and PBMC samples showed lower detection rates of 4.17% and 9.47%, respectively. The overall positivity for scrub typhus was 29.5%, highlighting the significant role of this pathogen in pediatric AES cases in endemic regions. The phylogenetic analysis of scrub typhus revealed many Gilliam-like strains. Conclusion ELISA for scrub typhus IgM has a high positivity rate in comparison to PCR for the detection of scrub typhus in children with AES.