This study aims to investigate the associations of O6-methylguanine-DNA methyltransferase (MGMT) genetic polymorphisms (Leu84Phe and Ile143Val) with temozolomide (TMZ) resistance and prognosis of patients with malignant gliomas. A total of 212 patients diagnosed with malignant gliomas were enrolled in this study as the case group. All of these patients took oral TMZ and were assigned into the TMZ-sensitive (complete response+partial response) and the TMZ-resistant (stable disease+progressive disease) groups based on the clinical response after chemotherapy. The polymerase chain reaction-restriction fragment length polymorphism was used to identify the gene polymorphism of Leu84Phe and Ile143Val. The survival time and survival outcomes of all the patients were obtained by follow-up. There were significant differences in the genotype and allele of Leu84Phe between the TMZ-sensitive and the TMZ-resistant groups. The CT, TT and CT+TT genotypes and the T allele of MGMT gene Leu84Phe may be associated with increasing TMZ resistance in patients with malignant gliomas. Logistic regression analysis showed that Leu84Phe of MGMT gene and pathological grade were independent risk factors for the increase of TMZ resistance in patients with malignant gliomas. Kaplan-Meier survival curve revealed that the average survival time of patients with the CT+TT and CC genotypes of Leu84Phe in the two groups was statistically significant. COX regression analysis showed that Leu84Phe, degree of resection and pathological grade were independent prognostic factors for patients with malignant gliomas. Our study demonstrates that Leu84Phe of MGMT gene might be a risk factor of TMZ resistance and poor prognosis of patients with malignant gliomas.