Precision oncology trials for pediatric cancers require rapid and accurate detection of genetic alterations. Tumor variant identification should interrogate the distinctive driver genes and more frequent copy number variants and gene fusions that are characteristics of pediatric tumors. Here, we evaluate tumor variant identification using whole genome sequencing (n = 12 samples) and two amplification-based next-generation sequencing assays (n = 28 samples), including one assay designed to rapidly assess common diagnostic, prognostic, and therapeutic biomarkers found in pediatric tumors. Variant identification by the three modalities was comparable when filtered for 151 pediatric driver genes. Across the 28 samples, the pediatric cancer-focused assay detected more tumor variants per sample (two-sided, P < .05), which improved the identification of potentially druggable events and matched pathway inhibitors. Overall, our data indicate that an assay designed to evaluate pediatric cancer-specific variants, including gene fusions, may improve the detection of target-agent pairs for precision oncology.