The purpose of the study was to investigate renal oxidative stress (OS) and its relationship with disease severity in patients with chronic kidney disease (CKD) using positron emission tomography coupled with magnetic resonance imaging (PET/MRI), employing 64Cu-diacetyl-bis(N4-methylthiosemicarbazonate) (64Cu-ATSM) as the PET tracer for OS imaging. Thirty patients with CKD (66.4 ± 8.2 y.o.) and seven healthy controls (HC) subjects (58.3 ± 3.8 y.o.) underwent 64Cu-ATSM PET/MRI. Participants were categorized into three groups based on their estimated glomerular filtration rate (eGFR): HC, mild CKD (stages 2-3a), and advanced CKD (stages 3b-5). All subjects underwent 30-min dynamic PET/MRI starting with the injection of 64Cu-ATSM to evaluate renal blood flow (RBF) and OS levels. RBF (mL/min/100g) images were calculated from the first 3 min PET data, and standardized uptake value (SUV) images were obtained from delayed frames of 15–30 min after injection. The 64Cu-ATSM SUV images were corrected using individual RBF images to estimate the OS levels of individual kidneys using the following equation: OS index = (SUV/RBF)x100. Significant correlation was observed between eGFR and RBF (r = 0.81, P < 0.001). RBF in patients with advanced CKD is significantly lower than that in HC (P < 0.001) and patients with mild CKD (P = 0.004). 64Cu-ATSM SUV did not differ significantly among the three groups (P = 0.171). 64Cu-ATSM SUVs did not correlate with creatinine in the HC subjects or in the patients with CKD. However, these values did correlate with eGFR (r = 0.33, P = 0.049) in all subjects, whereas the CKD patients showed no significant correlation. Following RBF correction, the OS index demonstrated significant correlations with creatinine (r = 0.75, P < 0.001), eGFR (r= -0.65, P < 0.001), and CKD stages (r = 0.57, P < 0.001) in all subjects. This preliminary study has revealed that 64Cu-ATSM PET may provide a reasonable estimate of renal OS reasonably in CKD patients noninvasively. Increased OS index values were correlated with the CKD stages and creatinine levels, suggesting that OS increases with the severity of renal dysfunction.