MSH6 (mutS homolog 6), one of the five key mismatch repair (MMR) genes, was found to play an important role in conferring resistance to alkylating agents-temozolomide (TMZ) in malignant glioma. This study aims to investigate whether genetic variations in MSH6 gene are associated with the survival outcomes in patients with malignant glioma. Each exon of the MSH6 gene was sequenced, and single nucleotide polymorphism (SNP) analysis was performed using 74 tumor tissues from glioblastoma multiforme (GBM) patients. Among these patients, 54 patients received radiotherapy plus TMZ treatment; 20 patients had radiotherapy only. The promoter methylation of O6-methylguanine methyltransferase (MGMT) was measured by methylation-specific polymerase chain reaction. Literature mining and related data collection were done with NCBI and PubMed databases. Of the 74 GBM patients, 50% (n = 37) harbored MSH6 G268A polymorphism, and no significant rates of other SNP or gene mutation across MSH6 exons were detected. The median overall survival (OS) was 15.6 months for who harbored the SNP and 12.6 months for SNP-negative patients (log-rank test: p = .324). The median OS for the MGMT promoter methylation group (n = 25) and nonmethylation group (n = 29) of the 54 GBM patients treated with TMZ was 21.3 and 8.9 months, respectively, (p = .002). In conclusion, we identified a high frequency of MSH6 G268A polymorphism in MSH6 gene, which did not have a notable influence on survival for the malignant glioma patients with/without TMZ treatment.