The repair protein O6-methylguanine-DNA methyltransferase (MGMT) is regulated epigenetically, mainly by the methylation of theMGMTpromoter.MGMTpromoter methylation status has emerged as a prognostic and predictive biomarker for patients with newly diagnosed glioblastoma (GBM). However, a strong negative correlation betweenMGMTpromoter methylation and MGMT protein expression cannot be applied as a rule for all GBM patients. In order to investigate if the DNA methylation status ofMGMTenhancers is associated withMGMTpromoter methylation, MGMT expression, and the overall survival (OS) of GBM patients, we established assays based on high-resolution melting analysis and pyrosequencing for one intragenic and three intergenicMGMTenhancers. For CpGs in an enhancer located 560 kb upstream of theMGMTpromoter, we found a significant negative correlation between the methylation status and MGMT protein levels of GBM samples expressing MGMT. The methylation status of CpGs in the intragenic enhancer (hs696) was strongly negatively correlated withMGMTpromoter methylation and was significantly higher in MGMT-expressing GBM samples than in MGMT-non-expressing GBM samples. Moreover, low methylation of CpGs 01–03 and CpGs 09–13 was associated with the longer OS of the GBM patients. Our findings indicate an association betweenMGMTenhancer methylation andMGMTpromoter methylation, MGMT protein expression, and/or OS.