Diffuse midline glioma (DMG; a subtype of pediatric high-grade glioma) is a fatal disease in children, due to the localization in critical structures of the central nervous system, its invasive nature, and limited treatment options. Molecularly, DMG with loss of histone 3 K27 trimethylation (mostly through the typical K27M-mutation in histone 3) have been relatively well characterized, however, no unambiguous Achilles' heel for targeted therapeutic approaches could be identified to date. This study integrates detailed molecular characteristics of pediatric DMGs with clinical data in a large, international cohort in order to contribute to a better understanding necessary for further development of therapeutic approaches. A total of 162 DMG tumors were analyzed within the INFORM registry from 01/2015 to 11/2023 using comprehensive molecular profiling (including exome, whole-genome and RNA next-generation sequencing approaches, complemented with DNA methylation analysis). Molecular results were correlated with clinical data of the respective patients including the treatment regimen applied and patients' outcomes. This well-defined cohort of histone 3 K27-altered DMG according to the current WHO classification showed typical molecular alterations for this entity, with differences in frequencies in specific subgroups. The presence of TP53 mutation and the absence of MAPK pathway alteration in the tumors were associated with worse outcomes. In a substantial proportion of patients, genetic alterations serving as targets for potential therapeutic approaches could be identified. This large, international, prospective DMG cohort combines comprehensive molecular characterization of the tumors with registry-level clinical data, thereby contributing to a better understanding of the underlying tumor biology, potential prognostic and predictive markers and the potential impact of targeted therapies.