Somatic mutations in histone H3 were identified in pediatric diffuse intrinsic pontine gliomas (DIPGs) and non-brainstem pediatric glioblastomas (non-BS-PGs). Whole genome sequencing of 7 DIPGs and matched germline DNA, along with targeted sequencing of 43 DIPGs and 36 non-BS-PGs, revealed that 78% of DIPGs and 22% of non-BS-PGs had p.K27M mutations in H3F3A or HIST1H3B, encoding histone H3.3 or H3.1. An additional 14% of non-BS-PGs had p.G34R mutations in H3F3A. These mutations were mutually exclusive and not present in other histone H3 genes. The mutations were somatic, as germline DNA was wild-type. The p.K27M mutation was found in seven of eight DIPG samples obtained prior to therapy, indicating it was not therapy-induced. No K27 or G34 mutations were found in other histone H3 genes. The mutations were not associated with chromosomal gain or amplification of other genes. The mutations occurred in the N-terminal tail of histone H3, which influences chromatin structure and function. These mutations may affect histone-DNA interactions, chromatin compaction, or interactions with other effectors. The mutations may impact epigenetic regulation, developmental gene regulation, or telomere stability. These mutations were not found in adult glioblastomas, suggesting a unique role in pediatric brain development. The mutations in H3F3A and HIST1H3B may provide a selective advantage in the context of developing brain. The findings highlight the importance of histone H3 mutations in pediatric gliomas and their potential impact on epigenetic regulation.Somatic mutations in histone H3 were identified in pediatric diffuse intrinsic pontine gliomas (DIPGs) and non-brainstem pediatric glioblastomas (non-BS-PGs). Whole genome sequencing of 7 DIPGs and matched germline DNA, along with targeted sequencing of 43 DIPGs and 36 non-BS-PGs, revealed that 78% of DIPGs and 22% of non-BS-PGs had p.K27M mutations in H3F3A or HIST1H3B, encoding histone H3.3 or H3.1. An additional 14% of non-BS-PGs had p.G34R mutations in H3F3A. These mutations were mutually exclusive and not present in other histone H3 genes. The mutations were somatic, as germline DNA was wild-type. The p.K27M mutation was found in seven of eight DIPG samples obtained prior to therapy, indicating it was not therapy-induced. No K27 or G34 mutations were found in other histone H3 genes. The mutations were not associated with chromosomal gain or amplification of other genes. The mutations occurred in the N-terminal tail of histone H3, which influences chromatin structure and function. These mutations may affect histone-DNA interactions, chromatin compaction, or interactions with other effectors. The mutations may impact epigenetic regulation, developmental gene regulation, or telomere stability. These mutations were not found in adult glioblastomas, suggesting a unique role in pediatric brain development. The mutations in H3F3A and HIST1H3B may provide a selective advantage in the context of developing brain. The findings highlight the importance of histone H3 mutations in pediatric gliomas and their potential impact on epigenetic regulation.