In radiation therapy (RT), staging, treatment planning, monitoring and evaluation of response are traditionally based on computed tomography (CT) and magnetic resonance imaging (MRI). These radiological investigations have the significant advantage to show the anatomy with a high resolution, being also called anatomical imaging. In recent years, so called biological imaging methods which visualize metabolic pathways have been developed. These methods offer complementary imaging of various aspects of tumour biology. To date, the most prominent biological imaging system in use is positron emission tomography (PET), whose diagnostic properties have clinically been evaluated for years. The aim of this review is to discuss the valences and implications of PET in RT. We will focus our evaluation on the following topics: the role of biological imaging for tumour tissue detection/delineation of the gross tumour volume (GTV) and for the visualization of heterogeneous tumour biology. We will discuss the role of fluorodeoxyglucose-PET in lung and head and neck cancer and the impact of amino acids (AA)-PET in target volume delineation of brain gliomas. Furthermore, we summarize the data of the literature about tumour hypoxia and proliferation visualized by PET. We conclude that, regarding treatment planning in radiotherapy, PET offers advantages in terms of tumour delineation and the description of biological processes. However, to define the real impact of biological imaging on clinical outcome after radiotherapy, further experimental, clinical and cost/benefit analyses are required.