Abstract Purpose: The methylation status of the O6-methylguanine DNA methyltransferase (MGMT) gene promoter is predictive for benefit from temozolomide in glioblastoma (GBM). A clinically optimized cutoff was sought allowing patient selection for therapy without temozolomide, while avoiding to withhold it from patients who may potentially benefit. Experimental Design: Quantitative MGMT methylation-specific PCR data were obtained for newly diagnosed patients with GBM screened or treated with standard radiotherapy and temozolomide in four randomized trials. The pooled dataset was randomly split into a training and test dataset. The unsupervised cutoff was obtained at a 50% probability to be (un)methylated. ROC analysis identified an optimal cutoff supervised by overall survival (OS). Results: For 4,041 patients valid MGMT results were obtained, whereof 1,725 were randomized. The unsupervised cutoff in the training dataset was 1.27 (log2[1,000 × (MGMT+1)/ACTB]), separating unmethylated and methylated patients. The optimal supervised cutoff for unmethylated patients was −0.28 (AUC = 0.61), classifying “truly unmethylated” (≤−0.28) and “gray zone” patients (>−0.28, ≤1.27), the latter comprising approximately 10% of cases. In contrast, for patients with MGMT methylation (>1.27) more methylation was not related to better outcome. Both methylated and gray zone patients performed significantly better for OS than truly unmethylated patients [HR = 0.35, 95% confidence interval (CI), 0.27–0.45, P < 0.0001; HR = 0.58, 95% CI, 0.43–0.78, P < 0.001], validated in the test dataset. The MGMT assay was highly reproducible upon retesting of 218 paired samples (R2 = 0.94). Conclusions: Low MGMT methylation (gray zone) may confer some sensitivity to temozolomide treatment, hence the lower safety margin should be considered for selecting patients with unmethylated GBM into trials omitting temozolomide.