As tumors represent one of the most common causes of death in developed countries, it is of utmost importance to understand the (molecular) mechanisms underlying tumor progression. On this basis, cancer diagnosis and therapy can be improved. Specific radioactive molecules can help in providing such information when detected by sensitive cameras, especially when this is done in a three-dimensional manner. Positron-emitting radioisotopes can be detected by positron emission tomography (PET) cameras. A range of molecular processes can be visualized by PET, depending on the radiotracer used. However, before novel tracers can be used in a clinical scenario, it is fundamental to thoroughly prove their specificity in in vitro experiments and also in animal models. And also tracers, which are already clinically used, may be further validated in preclinical studies. To this end, small animal models of cancer and also dedicated preclinical PET cameras have been developed. Preclinical PET, in combination with CT or MRI, has fundamentally contributed to the establishment of novel imaging, diagnostic, therapy and theranostic approaches in the clinics. Here, we provide an overview of the diverse applications and also the challenges of preclinical PET in the field of oncology.