OBJECTIVE Atherosclerosis remains the pathological basis of myocardial infarction and ischemic stroke. Early and accurate identification of plauqes is crucial to improve clinical outcomes of atherosclerosis patients. Our study aims to evaluate the potential value of fibroblast activation protein inhibitor (FAPI)-04 PET/CT in identifying plaques via a preclinical rabbit model of atherosclerosis. METHODS New Zealand white rabbits were fed high-fat diet (HFD), and randomly divided into the model group injured by the balloon, and the sham group only with incisions. Ultrasound was performed to detect plaques, and FAPI-avid was determined through Al18F-NOTA-FAPI-04 PET/CT. Mean standardized uptake values (SUVmean) in lesions were compared, and biodistribution of Al18F-NOTA-FAPI-04 and target-to-background ratios (TBRs) were calculated. Histological staining was performed to display arterial plaques, and autoradiography (ARG) was employed to measure the in vitro intensity of Al18F-NOTA-FAPI-04. At last, the correlation among FAP levels, plaque area, SUVmean values and fibrous cap thickness was assessed. RESULTS The rabbit carotid and abdominal atherosclerosis model was established. Al18F-NOTA-FAPI-04 showed a higher uptake in carotid plaques (SUVmean 1.32 ± 0.11) and abdominal plaques (SUVmean 0.73 ± 0.13) compared to corresponding controls (SUVmean 1.07 ± 0.06; 0.46 ± 0.03) (P < 0.05). Biodistribution analysis of Al18F-NOTA-FAPI-04 revealed that the bigger plaques were delineated with higher TBRs. Pathological staining showed the formation of arterial plaques, and ARG staining exhibited a higher intensity of Al18F-NOTA-FAPI-04 in the bigger plaques. Lastly, plaque area was found to be positively correlated to FAP expression and SUVmean, while FAP expression was negatively correlated to fibrous cap thickness of plaques. CONCLUSIONS We successfully achieve molecular imaging of fibroblast activation in atherosclerotic lesions of rabbits, suggesting Al18F-NOTA-FAPI-04 PET/CT may be a potentially valuable tool to identify plaques.