Diffuse midline glioma (DMG), H3 K27M-mutant occurs in both adult and pediatric populations. The characteristics of the two DMG groups were systematically explored in this study.  One-hundred-sixteen patients diagnosed with H3 K27M-mutant DMG in Beijing Tiantan Hospital from May 2016 to December 2018 were included. Patients were classified into adult group (n=57, 49.1%) and pediatric group (n=59, 50.9%). The clinical, radiological and molecular features were compared between the groups. Univariate and multivariate analyses were performed to identify prognostic factors. Compared with the adult group, pediatric patients had a younger age (8.9±4.1 vs. 35.1±11.8 years, P<0.001), a lower preoperative Karnofsky performance scale score (62.9±15.5 vs. 72.1±16.5, P=0.004), a lower rate of total resection (5.7% vs. 26.8%, P=0.009), a larger tumor size (4.4±0.9 vs. 3.9±1.5 cm, P=0.045), a higher Ki-67 index (63.0% vs. 37.8%, P=0.047), and higher rates of postoperative cranial nerve palsy (61.0% vs. 36.8%, P=0.009) and ataxia (45.8% vs. 26.3%, P=0.029). Adult DMG was predominantly located in the thalamus, while the predilection site for pediatric DMG was brainstem (P<0.001). Kaplan-Meier plot showed that the median survival of adult and pediatric DMG was 16.0 (9.7-22.3) months and 10.0 (8.3-11.7) months, respectively, which imparted a significant difference (P=0.008). Age at diagnosis, radiotherapy, motor deficit and hydrocephalus were confirmed as independent prognostic factors according to the multivariate analysis (P<0.05). Compared with adult patients, children with H3 K27M-mutant DMG confer distinct clinical, radiological and molecular characteristics, and have a dismal prognosis. Radiotherapy is an independent factor associated with prolonged survival. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.