<h3>Purpose/Objective(s)</h3> It is known that cancer stem cells contribute to disease progression and treatment resistance. However, the clinical value of stemness in glioblastoma (GBM) has not been fully elucidated. Here, we attempted to construct a stemness-related gene signature (SRGS) based on the expression profile of genes to predict the treatment efficacy of radiotherapy in GBM. <h3>Materials/Methods</h3> Transcriptome data and corresponding clinical information of GBM patients were extracted from the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) database. These stemness-related genes with both differentially expression and prognostic value were used to construct the stemness-related gene signature (SRGS). The performance of the SRGS was evaluated using Kaplan-Meier survival analysis, receiver operating characteristic (ROC) curves and correlation analysis of molecular characteristics, while the TCGA database was used for external validation. Next, we evaluated the predictive value of SRGS for treatment efficacy of radiotherapy in GBM. <h3>Results</h3> The prognostic gene signature was composed of nine genes, including ASPM, EPHA1, LIF, LOXL2, NAP1L2, OTP, PITX2, RDH10 and RUNX1. Patients were divided into high and low-risk groups according to the median risk score of SRGS. Patients in the high-risk group had significantly poor overall survival (OS) than that of patients in the low-risk group (1-year OS, 46.3% vs. 53.8%, P=0.018; 2-year OS, 17.2% vs. 34.2%, P=0.002; 3-year OS 6.7% vs. 26.2%, P<0.001). Meanwhile, the area under the ROC curves of the SRGS was 0.603, 0.724, and 0.763 for 1-, 2-, and 3-year OS in the CGGA cohort, and demonstrated stable consistency in the TCGA external validation. In the high-risk group, patients receiving radiotherapy had better OS than those without receiving radiotherapy (1-year OS, 58.0% vs. 23.8%, P=0.001; 2-year OS, 19.7% vs. 4.8%, P=0.016; 3-year OS, 9.9% vs. 0.0%, P<0.001). However, radiotherapy failed to improve OS in low-risk group, with no statistically significant differences in cohorts between radiotherapy and without radiotherapy at 1 year OS, 50.0% vs. 44.4%, P=0.396; 2-years OS, 33.0% vs. 22.4%, P=0.427; 3-years OS, 24.8% vs. 13.5%, P=0.397). Higher risk scores of SRGS were observed in patients with wild-type IDH1, unmethylated MGMT promoter and 1p/19q non-codeletion, respectively. Multivariate analysis found SRGS was an independently prognostic factor of OS for GBM. <h3>Conclusion</h3> We constructed and validated a novel stemness-related gene signature. Moreover, this gene signature could predict the treatment efficacy of radiotherapy for GBM.