Background: Gliomas regularly evade current therapies through primary and acquired resistance and the effect of temozolomide is mainly restricted to the subgroup of methylguanin-O6-methyltransferase promoter (MGMT) hypermethylated tumors. Further resistance markers and pathways against chemotherapy, radiotherapy and targeted agents are unknown, but will be important in targeted warehouse studies like NCT Neuro Master Match (N²M²). Methods: The diagnostic pipeline involves clinical and molecular analysis of WHO grade III (n = 116) and WHO grade IV (n = 400) glioma patient samples focusing on methylation profiles as well as a prospective feasibility study of real time multilayer molecular profiling on various levels in 43 WHO grade IV patients with an unmethylated MGMT promoter. Results:Isocitrate dehydrogenase (IDH) wildtype glioblastomas mainly show three different methylation clusters (RTKI, RTKII and mesenchymal). Samples of Cluster 1 (RTKII) had higher prevalence of MGMT methylation, TERT methylation and TERT promoter mutation. MGMT methylation is only prognostic in this cluster, likely to be linked to TERT status. TERT promoter mutation is further associated with changes in the global methylation profile. DNA damage response (DDR) gene methylation patterns show association with survival in grade IIII and IV glioma, including POLE4 linked to radiation response. The prospective N²M² pilot study demonstrates feasibility of complex molecular profiling within four weeks and identified high confidence markers for targeted therapy in 35% of the patients. Furthermore, a therapeutic algorithm is proposed to allocate patients with high grade gliomas to the best possible treatment based on molecular biomarkers. This algorithm is ready to be adjusted by continues input from targeted clinical and preclinical studies and in vivo CRISPR screens. Conclusions: Multilayer molecular biomarker analysis for high grade glioma is feasible and can be implemented in a therapeutic algorithm estimating the most promising therapy according to current knowledge. Specifically, the TERT-DDR axis may be involved in resistance to chemo- and radiotherapy in high-grade glioma.