Significance:Electron Paramagnetic Resonance imaging (EPRI) is a powerful technique capable of generating images of tissue oxygenation using exogenous paramagnetic probes such as trityl radicals and nitroxyl radicals. Using principles similar to Magnetic Resonance Imaging (MRI) with field gradients, the spatial distribution of the paramagnetic probecan be generated and from its spectral features, spatial maps of oxygen can be obtained from live objects. In this review, the two methods of signal acquisition, image formation/reconstruction will be described. The probes used and its application to study tumor physiology and monitor treatment response with chemotherapy drugs in mouse models of human cancer will be summarized.Recent Advances: By implementing phase encoding/Fourier reconstruction in EPRI in time-domain mode, the frequency contribution to the spatial resolution was avoided and improved images can be obtained. The highresolution EPRI revealed the pO2 change in tumor, which was useful to detect and evaluate the effects of various antitumor therapies. The coregistration with other imaging modalities provided a better understanding of hypoxia related alteration in physiology.Critical Issues: The high power of EPR irradiation and toxicity profile of radical probes are the main obstacles for clinical application. The improvement of pulse sequence may lower the risk.Future Directions:Pulsed EPR oximetry will be a powerfultool to research various disease involving hypoxia such as cancer, ischemic heart diseases, stroke, and diabetes. By optimizing radical probes, it can also be applied for various other purposes such as detecting local acid-base balance or oxidative stress