Objective To investigate the clinicopathological characteristics of pediatric H3 K27-altered diffuse midline gliomas (DMG). Methods The clinical and pathological data of children first diagnosed with H3 K27-altered DMG from September 2016 to July 2021 were retrospectively analyzed. Results A total of 95 patients (54 boys and 41 girls) were included, and the median age was 6.0 (5.0-9.0) years. Brainstem was the predilection site (82.1%). WHO histological grade 1-4 tumors were found in all cases and high-grade gliomas were found in most cases (65.3%). Immunohistochemical detection showed that H3 K27M (the 27th lysine of histone H3 changed to methionine) was positively expressed in all cases, and H3 K27Me3 (the trimethylation of the 27th lysine of histone H3) was decreased in 89.0% of the children. Mutant p53 (P53) was expressed in 55.6% of the children. The expression rate of isocitrate dehydrogenase 1 (IDH1) and loss expression rate of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) were low (8.5% and 18.6%, respectively). V-Raf murine sarcoma viral oncogene homolog B1 (BRAF) was positive in only 1 case (1.2%). Molecular tests showed that O-6-methylguanine-DNA methyltransferase (MGMT) methylation was detected in 8 cases (47.1%, 8/17), and BRAF variation was detected in 1 case (2.1%, 1/48). Conclusions The commonest location of H3 K27-altered DMG children is the brainstem, and the histological grade is mostly high. ATRX loss in pediatric cases is less common and had no relationship with the tumor location.