Objective: Cardiac autonomic neuropathy(CAN) is one of the serious complications of diabetes, which might lead to myocardial ischemia and myocardial infarction. The aim of this study is to evaluate the clinical application of myocardial perfusion imaging(MPI) in the diagnosis of myocardial ischemia in patients with CAN. Methods: Forty-three patients with type 2 diabetes were divided into 4 groups: normal function group(N=8), early phase group(N=9), mid phase group(N=16) and late phase group(N=10). Rest-delay MPI using 201Tl and Holter were performed in all patients. Results: 55.8%(24/43) of patients were found by MPI to have myocardial ischemia, with a total of 67 reversible ischemic segments and 18 irreversible ischemic segments. The incidence of myocardial ischemia diagnosed by MPI was revealed to have a proportionate upward trend with the deterioration of CAN. Holter indicated a similar result with a comparatively low sensitivity(32.6%). Conclusions: MPI using 201Tl was proved by the study to be superior to Holter in the diagnosis of myocardial ischemia in patients with CAN. Early intervention will be accessible in CAN patients by using MPI.