Renal oxygenation is essential for maintaining kidney function. Disruptions in oxygen delivery can lead to renal hypoxia, which can exacerbate kidney injury through multiple pathways, including inflammation, oxidative stress, and ischemia-reperfusion injury. Despite the recognized importance of oxygenation in renal pathology, non-invasive and reliable methods for assessing kidney oxygen levels are limited. Current techniques either lack sensitivity or involve invasive procedures, restricting their use in routine monitoring. Therefore, there is a pressing need for innovative approaches to assess renal oxygenation, particularly in kidney injury. This study evaluated Electron Paramagnetic Resonance (EPR)-based oxygen imaging using the paramagnetic tracer Ox071 to assess kidney oxygen levels in mice with cyclophosphamide-induced kidney injury. Urine pO2 was also assessed as a potential surrogate marker. EPR oximetry accurately measured kidney oxygen distribution, revealing a temporary increase in pO2 post-injury. Urine oximetry, however, did not reliably reflect changes in kidney oxygenation. Furthermore, EPR oximetry provided high-resolution spatial mapping of oxygen levels within the kidney, allowing for a detailed understanding of the impact of hypoxia on renal tissue. EPR oximetry is a promising, non-invasive tool for monitoring renal oxygenation, offering high-resolution mapping and longitudinal assessment. Its ability to provide detailed information about oxygen distribution within the kidney makes it a valuable tool for studying the pathophysiology of renal diseases and for developing novel therapeutic strategies.