BACKGROUND: Up to 30% of pediatric glioblastomas (GB) demonstrate mutations in H3F3A, a gene variant encoding histone H3.3. Global reduction in H3K27me3 is associated with H3F3A K27M mutation. METHODS: We evaluated the utility of histopathological detection of this reduction in H3K27me3 as a prognostic biomarker. H3K27me3 expression was established by immunohistochemistry in 160 (118 pediatric and 42 adult) brain tumors of various subtypes and grades and 45 non-neoplastic reactive brain tissues. RESULTS: Global reduction in H3K27me3 was specific to GB cases with the H3F3A K27M mutantion, and was not observed in all other cases examined. In addition, H3F3A K27M mutant GB with reduced H3K27me3 exhibited a poor prognosis compared to H3F3A wild type tumors with preserved H3K27me3 (Log-rank (Mantel-Cox) test hazards ratio = 2.9, p = 0.0021). CONCLUSIONS: Global reduction in H3K27me3 is a histopathologically detectable surrogate for H3F3A K27M mutant GB that defines a poor prognosis subset of pediatric GB. SECONDARY CATEGORY: Pediatrics.