The development of positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging continues to grow due to the ability of these techniques to allow the non-invasive in vivo visualisation of biological processes at the molecular and cellular levels. As well as finding application for the diagnosis of disease, these techniques have also been used in the drug discovery process. Crucial to the growth of these techniques is the continued development of molecular probes that can bind to the target biological receptor with high selectivity. This tutorial review describes the use of PET and SPECT for molecular imaging and highlights key strategies for the development of molecular probes for the imaging of both cancer and neurological diseases.