External beam radiation therapy procedures have, until recently, been planned almost exclusively using anatomic imaging methods. Molecular imaging using hybrid positron emission tomography (PET)/computed tomography scanning or single-photon emission computed tomography (SPECT) imaging has provided new insights into the precise location of tumors (staging) and the extent and character of the biologically active tumor volume (BTV) and has provided differential response information during and after therapy. In addition to the commonly used radiotracer 18F-fluoro- 2-deoxyD-glucose (FDG), additional radiopharmaceuticals are being explored to image major physiological processes as well as tumor biological properties, such as hypoxia, proliferation, amino acid accumulation, apoptosis, and receptor expression, providing the potential to target or boost the radiation dose to a biologically relevant region within a tumor, such as the most hypoxic or most proliferative area. Imaging using SPECT agents has furthered the possibility of limiting dose to functional normal tissues. PET can also portray the distribution of particle therapy by displaying activated species in situ. With both PET and SPECT imaging, fundamental physical issues of limited spatial resolution relative to the biological process, partial volume effects for quantification of small volumes, image misregistration, motion, and edge delineation must be carefully considered and can differ by agent or the method applied. Molecular imaging-guided radiation therapy (MIGRT) is a rapidly evolving and promising area of investigation and clinical translation. As MIGRT evolves, evidence must continue to be gathered to support improved clinical outcomes using MIGRT versus purely anatomic approaches.