Understanding the (molecular) mechanisms underlying tumor progression is fundamental for developing and improving cancer diagnosis and therapy. Positron emission tomography (PET) is a method to non-invasively and longitudinally provide such information. Depending on the radioactive tracer employed, a range of molecular processes can be visualized. Preclinical PET has fundamentally contributed to the establishment of novel imaging, diagnostic, and therapy approaches in the clinical situation. It is a valuable tool to corroborate in vivo imaging findings with conventional ex vivo tissue analysis. Here, we provide an overview of challenges and applications of preclinical PET in the field of oncology.