Simple Summary: Pediatric high-grade gliomas (pHGGs) are highly aggressive tumors with a dismal prognosis despite multimodal therapy including surgery, radiation therapy and chemotherapy, underscoring the urgent need to develop novel therapeutic strategies. During tumor development, cells achieve immortality by activating telomere maintenance mechanisms. Alternative lengthening of telomere (ALT) is an important mechanism for maintaining telomere length and cell proliferation in tumor cells. However, the molecular pathway and prognostic significance of ALT activation in pHGGs are poorly understood. Here, we report the heterogeneity of telomere maintenance mechanisms and their association with genetic alterations with the presence of both ALT and telomerase activation in some tumors. These findings are particularly important for the future development of novel therapeutic strategies targeting ALT and telomerase in pHGGs. Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine glioma (DIPG), are highly aggressive tumors with dismal prognoses despite multimodal therapy including surgery, radiation therapy, and chemotherapy. To achieve cellular immortality cancer cells must overcome replicative senescence and apoptosis by activating telomere maintenance mechanisms (TMMs) through the reactivation of telomerase activity or using alternative lengthening of telomere (ALT) pathways. Although the ALT phenotype is more prevalent in pHGGs compared to adult HGGs, the molecular pathway and the prognostic significance of ALT activation are not well understood in pHGGs. Here, we report the heterogeneity of TMM in pHGGs and their association with genetic alterations. Additionally, we show that sensitivity to the protein kinase ataxia telangiectasia- and RAD3-related protein (ATR) inhibitor and the ATR downstream target CHK1 is not specific to pHGG ALT-positive cells. Together, these findings underscore the need for novel therapeutic strategies to target ALT in pHGG tumors. [ABSTRACT FROM AUTHOR]