BACKGROUND: Identifying the cause of infection is important for clinical management and public health decisions, including vaccination strategies. In low-resource settings, causes of fever are often not identified. In this study, molecular testing panels were used to identify the causes of pediatric fever in the Kathmandu Valley, Nepal. A dengue fever outbreak facilitated the investigation of dengue diagnostics. METHODS: Children under 14 years of age were recruited to this prospective cohort study at Patan Hospital, Nepal. Clinical data and routine diagnostics were used to classify cases, including nonstructural protein 1 (NS1) antigen testing for dengue. Additional molecular diagnostics were performed on blood (12 viral, 26 bacterial and 6 fungal targets) and respiratory samples (17 viral and 3 bacterial targets). RESULTS: From September 1, 2021, to April 19, 2023, 565 children were enrolled, median age 3 (interquartile-range 1-7) years. Pathogens identified included dengue virus (n = 101), respiratory syncytial virus (n = 30), influenza (n = 25), typhoidal Salmonella spp. (n = 7) and Neisseria meningitidis (n = 2). During the dengue outbreak, dengue polymerase chain reaction (PCR) and NS1 positivity rates were both high early in dengue disease, but if >3 days of symptoms, PCR positivity rates declined (10.3%) while NS1 positivity remained high into the second week of illness (80%). CONCLUSIONS: This prospective cohort study is the most comprehensive effort to date to describe the causes of pediatric fever in the Kathmandu Valley, Nepal. The United States Centers for Disease Control and Prevention recommends dengue PCR or NS1 antigen testing during the first 7 days of dengue fever. Our data indicate that PCR positivity declines after 3 days of symptoms, resulting in missed cases when relying solely on PCR.