Gliomas are the most common types of brain tumors, which invariably lead to death over months or years. Before new and potentially more effective treatment strategies, such as gene therapy, can be effectively introduced into clinical application the following goals must be reached: (1) the determination of localization, extent and metabolic activity of the glioma; (2) the assessment of functional changes within the surrounding brain tissue; (3) the identification of genetic changes on the molecular level leading to disease; and in addition (4) a detailed non-invasive analysis of both endogenous and exogenous gene expression in animal models and in the clinical setting. Non-invasive imaging of endogenous gene expression by means of positron emission tomography (PET) may reveal insight into the molecular basis of pathogenesis and metabolic activity of the glioma and the extent of treatment response. When exogenous genes are introduced to serve for a therapeutic function, PET imaging techniques may reveal the assessment of the location, magnitude and duration of therapeutic gene expression and its relation to the therapeutic effect. Here, we review the main principles of PET imaging and its key roles in neurooncology research.