Nuclear medicine uses radioactive probes, commonly referred to as radiotracers, for the diagnosis and treatment of diseases. As of today, single photon emission computed tomography (SPECT) has revolutionized the field of nuclear imaging and is one of the most important medical imaging methods providing functional information about physiologic and pathologic processes in biologic systems. In contrast to other imaging modalities used in clinical diagnosis, SPECT reveals information based on the spatial concentration of injected radiopharmaceutics. SPECT functional imaging provides increased sensitivity, improved contrast, reduced structural noise, and precise localization of defects. Functional imaging, however, has low specificity in distinctly different pathologies (e.g., degenerative, inflammatory, or malignant bone lesions) and limited spatial and temporal resolution. To overcome this disadvantage, highly specific probes (e.g., receptor imaging) have been developed for targeted clinical imaging. SPECT has found its way into most clinical scenarios in patients with endocrine tumors, neuroendocrine tumors, lung cancers, brain tumors, lymphoma, prostate cancer, and malignant bone lesions. Furthermore, hybrid SPECT/CT imaging is especially suited to support the increasing applications of minimally invasive surgery, as well as to precisely define the diagnostic and prognostic profile of cardiovascular patients. This chapter details the historical origins of emission tomography, basic principles of SPECT imaging, the current status of SPECT instrumentation and imaging, and a broad spectrum of clinical applications in the diagnosis of malignant diseases such as cancer.