Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor with poor prognosis and high potential of dispersion to other brain tissues in adult. Effective and modern choices of treatment including chemotherapy with alkylating agents marginally extend survival of GBM. However, alkylating agents can lead to highly harmful mismatch during DNA replication causing apoptosis and cell death. Accordingly, O6-Methylguanine-DNA methyltransferase (MGMT) removes alkyl adducts, thereby causing resistance to alkylating drugs. Single-Nucleotide Polymorphisms (SNPs) in MGMT promoter region may play a role in the regulation of MGMT expression and prediction of glioma development risk. In order to evaluate the clinical significance of rs1625649 SNP in the MGMT promoter region of glioblastoma, genomic DNA from a series of 54 patients with GBM and 50 healthy individuals in Iranian population were collected for tetra ARMS PCR amplification. None of the "A" or "C" alleles were associated with tumor occurrence, the "AA" genotype was more frequent in healthy subjects, and the "AC" genotype was 4.6 times more common in patients with GBM. The longest survival time was observed in the "CC" genotype; however, this difference was not statistically significant. On the other hand, homozygous rs1625649 (AA genotype) was significantly associated with a better survival than the cases with heterozygous rs1625649 (CA genotype) or wild type rs1625649 (CC genotype), predicting better response to temozolomide-based chemotherapy.