Abstract Background Recent studies have identified that glioblastoma IDH-wildtype (GBM IDH-WT) might be comprised of molecular subgroups with distinct prognoses. Therefore, we investigated the correlation between genetic alterations and survival in 282 GBM IDH-WT patients, to identify subgroups with distinct outcomes. Methods We reviewed characteristics of GBM IDH-WT (2009–2019) patients analyzed by next-generation sequencing interrogating 205 genes and 26 rearrangements. Progression-free survival (PFS) and overall survival (OS) were evaluated with the log-rank test and Cox regression models. We validated our results utilizing data from cBioPortal (MSK-IMPACT dataset). Results Multivariable analysis of GBM IDH-WT revealed that treatment with chemoradiation and RB1-mutant status correlated with improved PFS (hazard ratio [HR] 0.25, P < .001 and HR 0.47, P = .002) and OS (HR 0.24, P < .001 and HR 0.49, P = .016). In addition, younger age (<55 years) was associated with improved OS. Karnofsky performance status less than 80 (HR 1.44, P = .024) and KDR amplification (HR 2.51, P = .008) were predictors of worse OS. KDR-amplified patients harbored coexisting PDGFRA and KIT amplification (P < .001) and TP53 mutations (P = .04). RB1-mutant patients had less frequent CDKN2A/B and EGFR alterations (P < .001). Conversely, RB1-mutant patients had more frequent TP53 (P < .001) and SETD2 (P = .006) mutations. Analysis of the MSK-IMPACT dataset (n = 551) validated the association between RB1 mutations and improved PFS (11.0 vs 8.7 months, P = .009) and OS (34.7 vs 21.7 months, P = .016). Conclusions RB1-mutant GBM IDH-WT is a molecular subgroup with improved PFS and OS. Meanwhile, 4q12 amplification (KDR/PDGFRA/KIT) denoted patients with worse OS. Identifying subgroups of GBM IDH-WT with distinct survival is important for optimal clinical trial design, incorporation of targeted therapies, and personalized neuro-oncological care.