Evidence suggests hyperglycemia is associated with worse outcomes in glioblastoma (GB). This study aims to confirm the association between glycemia during radiotherapy (RT) and temozolomide (TMZ) treatment and overall survival (OS) in patients with newly diagnosed GB. This retrospective study included GB patients treated with RT and TMZ from 2004 to 2011, randomly divided into independent derivation and validation datasets. Time-weighted mean (TWM) glucose and dexamethasone dose were collected from start of RT to 4 weeks after RT. Univariate (UVA) and multivariable (MVA) analyses investigated the association of TWM glucose and other prognostic factors with overall survival (OS). In total, 393 patients with median follow-up of 14 months were analyzed. In the derivation set (n = 196) the median OS was 15 months and median TWM glucose was 6.3 mmol/L. For patients with a TWM glucose ≤6.3 and >6.3 mmol/L, median OS was 16 and 13 months, respectively (p = 0.03). On UVA, TWM glucose, TWM dexamethasone, age, extent of surgery, and performance status were associated with OS. On MVA, TWM glucose remained an independent predictor of OS (p = 0.03) along with TWM dexamethasone, age, and surgery. The validation set (n = 197), with similar baseline characteristics, confirmed that TWM glucose ≤6.3 mmol/L was independently associated with longer OS (p = 0.005). This study demonstrates and validates that glycemia is an independent predictor for survival in GB patients treated with RT and TMZ.