Due to the shortage of kidneys donated for transplantation, surgeons are forced to use the organs with an elevated risk of poor function or even failure. Although the existing methods for pre-transplant quality evaluation have been validated over decades in population cohort studies across the world, new methods are needed as long as delayed graft function or failure in a kidney transplant occurs. In this study, we explored the potential of utilizing photoacoustic (PA) imaging during normothermic machine perfusion (NMP) as a means of evaluating kidney quality. We closely monitored twenty-two porcine kidneys using 3D PA imaging during a two-hour NMP session. Based on biochemical analyses of perfusate and produced urine, the kidneys were categorized into ‘non-functional’ and ‘functional’ groups. Our primary focus was to quantify oxygenation (sO₂) within the kidney cortical layer of depths 2 mm, 4 mm, and 6 mm using two-wavelength PA imaging. Next, receiver operating characteristic (ROC) analysis was performed to determine an optimal cortical layer depth and time point for the quantification of sO₂ to discriminate between functional and non-functional organs. Finally, for each depth, we assessed the correlation between sO₂ and creatinine clearance (CrCl), oxygen consumption (VO₂), and renal blood flow (RBF). We found that hypoxia of the renal cortex is associated with poor renal function. In addition, the determination of sO₂ within the 2 mm depth of the renal cortex after 30 min of NMP effectively distinguishes between functional and non-functional kidneys. The non-functional kidneys can be detected with the sensitivity and specificity of 80% and 85% respectively, using the cut-off point of sO₂ < 39%. Oxygenation significantly correlates with RBF and VO₂ in all kidneys. In functional kidneys, sO₂ correlated with CrCl, which is not the case for non-functional kidneys. We conclude that the presented technique has a high potential for supporting organ selection for kidney transplantation.