Background: Bevacizumab remains the standard second line therapy in glioblastoma (GBM) with reported improved progression free survival, but not overall survival. We investigated a large clinico-genomic database of GBM patients treated with bevacizumab for molecular alterations associated with treatment outcome. Methods: Molecular profiles of GBM were tested by next-generation sequencing (NGS) of DNA (592 genes, NextSeq or whole-exome sequencing, Novaseq) and RNA (whole transcriptome sequencing, NovaSeq) at Caris Life Sciences (Phoenix, AZ). Gene amplification was determined by NGS with a threshold of > = 6 copies. Real-world survival information was obtained from insurance claims data. Time-on-treatment (TOT) of bevacizumab was calculated from start to finish of treatment while post-bevacizumab overall survival (bevOS) from start of bevacizumab to last day of contact. Short-term (ST) and long-term (LT) responders were defined as those with TOT ≤6 months and ≥1 year, respectively. Kaplan-Meier estimates were calculated and significance was determined as p values of < 0.05. For molecular comparisons, Fisher’s exact tests and Mann-Whiney U were used when appropriate. Results: Among the total of 383 ST and 107 LT patients identified, no significant difference in gender or age were seen. The LT cohort had significantly longer bevOS compared to the ST cohort (HR 0.27, [95% CI: 0.28-0.35], p < 0.0001). Interestingly, CDK4 amplification was seen in 23% (18/78) of LT patients but only 7% (21/300, p = 0.0002) of ST. When investigating the total cohort of 498 GBM treated with bevacizumab, CDK4 amplified patients demonstrated significantly improved TOT compared to non-amplified (HR: 0.639, [0.48-0.85, p = 0.002]). Conversely, the ST cohort had significantly more EGFR amplifications than the LT cohort (43%, 130/300 vs. 19%, 15/79, p < 0.0001). In the total bevacizumab-treated cohort, EGFR-amplified GBM had significantly worse TOT on bevacizumab than non-EGFR amplified GBM (HR 1.387, [1.15-1.67], p < 0.001). No associations were seen in other alterations explored including PIK3CA mutation (HR = 0.81 [0.587-1.113], p = 0.2), EGFR mutation (HR: 1.209, [0.977-1.495], p = 0.1) or RAF1 (HR: 0.647, [0.318-1.317], p = 0.2). Conclusions: Using a large clinical genomic database with GBM subjected to comprehensive molecular profiling, we demonstrated that amplification of CDK4 and EGFR were associated with long-term and short-term responses to bevacizumab, respectively. This warrants further investigation in independent cohorts controlled for age and other prognostic factors. If confirmed, a genetic basis for treatment optimization may provide meaningful clinical outcomes.