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This study examined pediatric tuberculosis (TB) cases in Osaka City from 2008-2023, focusing on diagnostic methods and molecular epidemiology using variable number of tandem repeats (VNTR) analysis and whole-genome sequencing (WGS). Among 29 children aged 0-14 years (notification rate: 0.04 per 100,000), three (10.3%) were born abroad, and five (17.2%) were born in Japan with source cases from foreign countries. Source cases were identified for 20 children (69.0%); 18 of these (90.0%) had smear-positive pulmonary TB. Seventeen children (58.6%) were identified through contact investigations, highlighting the crucial role of contact tracing and early detection. Diagnosing pediatric TB was challenging: 15 children (51.7%) were asymptomatic and 17 (58.6%) had culture-negative results. To enhance diagnostic accuracy, interferon-gamma release assays, chest computed tomography, and gastric aspirate examination for acid-fast bacilli are recommended. In pediatric TB cases with identified source patients, VNTR clusters were confirmed by WGS to be closely related. Among children without identified sources, four formed VNTR clusters, and WGS of one representative case revealed no epidemiological links. These findings highlight the need to integrate molecular and epidemiological investigations to clarify transmission routes. Overall, this research underscores the need to integrate diagnostic and epidemiological approaches for effective control of pediatric TB.