O6-methylguanine-methyltransferase (MGMT) promoter methylation status has prognostic and, in the subpopulation of elderly patients, predictive value in newly diagnosed glioblastoma. Therefore, knowledge of the MGMT promoter methylation status is important for clinical decision-making. So far, MGMT testing has been limited by the lack of a robust test with sufficiently high analytical performance. Recently, one of several available pyrosequencing protocols has been shown to be an accurate and robust method for MGMT testing in an intra- and interlaboratory ring trial. However, some uncertainties remain with regard to methodological issues, cut-off definitions, and optimal use in the clinical setting. In this article, we highlight and discuss several of these open questions. The main unresolved issues are the definition of the most relevant CpG sites to analyze for clinical purposes and the determination of a cut-off value for dichotomization of quantitative MGMT pyrosequencing results into "MGMT methylated" and "MGMT unmethylated" patient subgroups as a basis for further treatment decisions.