Background: The treatment for ampullary cancer is pancreatoduodenectomy or local ampullectomy. However, effective methods for the preoperative investigation of hilar biliary invasion in ampullary cancer patients have not yet been identified. In this study, we aimed to determine an appropriate method for investigating hilar biliary invasion of ampullary cancer.Methods: Among 43 ampullary cancer patients, 34 underwent endoscopic treatment (n = 9) or surgery (n = 25). Imaging findings (thickening and enhancement of the bile duct wall on contrast-enhanced CT (CECT), irregularity on endoscopic retrograde cholangiography (ERC), thickening of the entire bile duct wall on intraductal ultrasonography (IDUS), and partial thickening of the bile duct wall on IDUS) and biliary biopsy results were compared with respect to their ability to diagnose hilar biliary invasion of ampullary cancer.Results: Hilar invasion was not observed in every patient. Among the patients who did not undergo biliary stent insertion, the combination of partial thickening of the bile duct wall on IDUS and biliary biopsy showed the highest accuracy (100%) for diagnosing biliary invasion. However, each imaging finding and biliary biopsy yielded some false positive results.Conclusions: Although some false positive results were obtained with each method, the combination of partial thickening of the bile duct wall on IDUS and biliary biopsy was useful for diagnosing hilar biliary invasion of ampullary cancer. However, hilar invasion of ampullary cancer is rare; therefore, the investigation of hilar biliary invasion of ampullary cancer might be unnecessary.Trial registration: not applicable