We have determined O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status by methylation-specific polymerase chain reaction (MSP) in 22 paraffin-embedded specimens of glioblastoma multiforme. A MGMT methylation-specific high resolution melting (HRM) assay was performed to compare the methylation levels of the tumorous and non-tumorous portions of each sample, which were selectively collected using a microdissection technique. MGMT methylation was detected in 10 patients using MSP, while 8 patients had both methylated and unmethylated MGMT promoters. HRM assays showed that there was no difference in the level of methylation between tumorous and non-tumorous portions of each sample. In patients with MSP-positive tumors, the overall survival (median, 22 months) was longer as compared to those with MSP-negative tumors (median, 14 months). A correlation between the methylation status of the MGMT promoter and MGMT protein expression was observed in 12 samples. This study demonstrates that MGMT methylation is not restricted to glioblastoma cells. Additionally, methylation-specific HRM is a feasible approach that can be readily applied to the methylation analysis of MGMT. A further study will be needed to determine the dynamic change of MGMT methylation in the tumor environment.