Background: Gliomas are the most common type of primary brain tumors with underlying molecular heterogeneity contributing to differential treatment response. Our retrospective study was designed to interrogate biomarkers from a large cohort of glioma patients to identify alterations with therapeutic implications. Methods: 871 glioma tumor samples (79% WHO grade IV glioblastoma, GBM) were analyzed with a multi-platform approach including sequencing, IHC, FISH/CISH and methylation assay to investigate actionable biomarker aberrations. Retrospective data analysis was performed on the complete cohort and molecular subgroups of patients. Results: In the 871 patient samples, mutations in 27 genes were seen. Both common TP53 (39%), IDH1 (22%), PTEN (13%) and previously unreported mutations in gliomas were observed, including JAK3, SMO and ABL1. Co-mutation of 2 or more genes occurred in 37% of cases. TP53 mutation was suggestive of genetic instability and was frequently associated with other concurrent mutations (p=0.0006). IDH1 mutations were associated with MGMT promoter methylation, low expression of TS, RRM1 and TOP2A (p from <0.0001 to 0.0036), suggesting different responses to temozolomide, fluoropyrimidine, gemcitabine and etoposide. IDH1 mutation was also associated with TP53 mutation; whereas wild type IDH1 was associated with PTEN mutation (p=0.0309) and showed some association with EGFR mutations (p=0.0543). Distinct biomarker profiles by IHC, FISH and sequencing were also observed when comparing GBM to grade II/III gliomas, suggesting different biology from GBM and thus different treatment implications. 20 GBM patients were identified with pre and post treatment analyses performed (comparative analysis ongoing). Conclusions: Gliomas exhibit a high degree of molecular heterogeneity as revealed by multi-platform profiling. IDH1 mutation identifies molecular subgroups of patients with different responses to therapeutic agents; while TP53 mutation suggests increased genetic instability. These results highlight the benefits of profiling in consideration of treatment options for glioma patients.