The correlation of pediatric high-grade gliomas with cancer predisposition syndromes (CPS) is poorly defined in the literature and next generation sequencing (NGS) is increasingly being used in support of diagnosis. We retrospectively analyzed NGS data from 77 consecutive children diagnosed with different types of pediatric high-grade glial tumors to identify the presence of germline variants predisposing to cancer. Sixty-four germline variants were detected in 44 cases (57.1%). Among all 64 variants, there were 6 (9.4%) pathogenic (P) variants, 8 (12.5%) likely pathogenic (LP), 40 (62.5%) variants of uncertain significance (VUS) and 10 (15.6%) likely benign and benign (LB/B). Finally, P/LP variants in CPS genes related were found in 13 children (16.9%). The distribution of variants was not significantly different in subgroups; however, the highest rate of P (80%) was observed in diffuse midline gliomas (DMG). Globally, P/LP were associated with lower overall-survival (59.2%) compared to VUS (74.1%) and no germline mutation/LB/B (80.9%). Our data represent the largest available NGS investigation in children with high-grade gliomas and suggest a potential prognostic disadvantage of P/LP compared to VUS and no mutation/LB.