Glioblastoma multiforme (GBM) (grade IV astrocytoma) has been assumed to be the most fatal type of glioma with low survival and high recurrence rates, even after prompt surgical removal and aggressive courses of treatment. Transcriptional reprogramming to stem cell-like state could explain some of the deregulated molecular signatures in GBM disease. The present study aimed to quantify the expression profiling of longevity-related transcriptional factors SOX2, OCT3/4, and NANOG to evaluate their diagnostic and performance values in high-grade gliomas. Forty-four specimens were obtained from glioblastoma patients (10 females and 34 males). Quantitative real-time polymerase chain reaction was applied for relative gene expression quantification. In silico network analysis was executed. NANOG and OCT3/4 mRNA expression levels were significantly downregulated while that of SOX2 was upregulated in cancer compared to noncancer tissues. Receiver operating characteristic curve analysis showed high diagnostic performance of NANOG and OCT3/4 than SOX2. However, the aberrant expressions of the genes studied were not associated with the prognostic variables in the current population. In conclusion, the current study highlighted the aberrant expression of certain longevity-associated transcription factors in glioblastoma multiforme which may direct the attention towards new strategies in the treatment of such lethal disease.