Objective To establish a method for quantitative determination of O⁶-methylguanine-DNA methyltransferase (MGMT) promoter methylation in gliomas using methylation-sensitive high-resolution melting (MS-HRM) curve, which is used as a guide to postoperative chemotherapy and prognostic prediction in patients with gliomas.Methods Glioma tissues from 37 patients were randomly selected from the specimen bank. DNA was extracted and modified by methylation. The MGMT promoter methylation level was determined by MS-HRM and methylation-specific PCR (MSP).Results According to the results of MSP, the number of specimen with partial methylation reached 29 (78.4%), which was significantly larger than that of specimen with full methylation (13.5%) or no methylation (8.1%). The results of MS-HRM analysis demonstrated that 14 specimen (37.8%) had their MGMT promoter methylation levels ranging between 10% and 25%, and 17 specimen (49.5%) between 25% and 50%.Conclusions This study successfully establishes a method for determination of MGMT promoter methylation in gliomas using MS-HRM. This method is highly specific, sensitive, and repeatable, which holds promise for clinical high-throughput quantitative determination of MGMT promoter methylation and a guide to personalized medication.