ASP5354 was recently developed as a near-infrared fluorescence (NIRF) contrast agent for intraoperative ureteral identification, and its use has been evaluated in healthy animals. However, the utilization of ASP5354 for ureteral identification has not been evaluated in animals with renal injury. In this study, we assessed the application of ASP5354 for ureteral imaging using rat models of gentamicin-induced mild, moderate, and severe acute kidney injury (AKI), using a clinically available NIRF detection system. NIRF was detected in the abdominal cavity and ureters after laparotomy, and the efficiency of ASP5354 was evaluated based on the NIRF signal intensity over 60 min. After the intravenous injection of ASP5354 into rats with mild or moderate AKI, the ureters were clearly imaged at a high ratio of NIRF intensity in the ureter to that in the tissues around the ureter. Six days after intravenous injection, the use of ASP5354 in rats with moderate AKI did not affect the biochemical kidney functions or histopathological conditions of the kidney tissues, as compared to those with no injection of ASP5354. In rats with severe AKI, ureteral imaging was not effective due to the relatively strong NIRF expression in the tissues around the ureters. These data indicate that ASP5354 holds potential as a contrast agent for intraoperative ureteral identification in patients with limited renal injury.