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Background: Nucleotide-specific 5-hydroxymethylcytosine (5hmC) remains understudied in pediatric central nervous system (CNS) tumors. 5hmC is abundant in the brain, and alterations to 5hmC in adult CNS tumors have been reported. However, traditional approaches to measure DNA methylation do not distinguish between 5-methylcytosine (5mC) and its oxidized counterpart 5hmC, including those used to build CNS tumor DNA methylation classification systems. We measured 5hmC and 5mC epigenome-wide at nucleotide resolution in glioma, ependymoma, and embryonal tumors from children, as well as control pediatric brain tissues using tandem bisulfite and oxidative bisulfite treatments followed by hybridization to the Illumina Methylation EPIC Array that interrogates over 860,000 CpG loci. Results: Linear mixed effects models adjusted for age and sex tested the CpG-specific differences in 5hmC between tumor and non-tumor samples, as well as between tumor subtypes. Results from model-based clustering of tumors was used to test the relation of cluster membership with patient survival through multivariable Cox proportional hazards regression. We also assessed the robustness of multiple epigenetic CNS tumor classification methods to 5mC-specific data in both pediatric and adult CNS tumors. Compared to non-tumor samples, tumors were hypohydroxymethylated across the epigenome and tumor 5hmC localized to regulatory elements crucial to cell identity, including transcription factor binding sites and super-enhancers. Differentially hydroxymethylated loci among tumor subtypes tended to be hypermethylated and disproportionally found in CTCF binding sites and genes related to posttranscriptional RNA regulation, such as DICER1. Model-based clustering results indicated that patients with low 5hmC patterns have poorer overall survival and increased risk of recurrence. Our results suggest 5mC-specific data from OxBS-treated samples impacts methylation-based tumor classification systems giving new opportunities for further refinement of classifiers for both pediatric and adult tumors. Conclusions: We identified that 5hmC localizes to super-enhancers, and genes commonly implicated in pediatric CNS tumors were differentially hypohydroxymethylated. We demonstrated that distinguishing methylation and hydroxymethylation is critical in identifying tumor-related epigenetic changes. These results have implications for patient prognostication, considerations of epigenetic therapy in CNS tumors, and for emerging molecular neuropathology classification approaches. [ABSTRACT FROM AUTHOR]