To systematically investigate the physiological distribution and benign lesion incidental uptake of Al18F-NOTA-FAPI-04 (18F-FAPI) in cancer patients to establish the normal uptake range in relevant organs and lesions.Twenty patients who underwent 18F-FAPI PET/CT imaging were retrospectively assessed. Organ and benign lesion tracer uptake was quantified based on standardized uptake values (SUVmax and SUVmean). We compared the variation in tracer uptake in certain organs between men and women, analyzed the possible reasons for diffuse uptake in the thyroid, and assessed tracer uptake variations in the uterus in different menstrual cycle phases. Incidental tracer uptake in benign lesions was also assessed.Physiological 18F-FAPI uptake was observed in the urinary tract, biliary tract system, submandibular glands, pancreas, thyroid, uterus, intestine, prostate gland, parotid gland, myocardium, kidney cortex, and muscles, but not the brain, lungs, liver, spleen, colon, and breasts. The SUVmean for each organ was similar for women and men (all P > 0.05). Diffuse tracer uptake in the thyroid was caused by normal thyroid or thyroiditis; there were no statistically significant differences between them (SUVmax: t = -1.3, P = 0.25; SUVmean: t = -1.1, P = 0.31). There was a significant difference for uterus uptake among different menstrual cycle phases (SUVmax: F = 5.08, P = 0.04; SUVmean: F = 5.19, P = 0.04). Incidental benign lesion tracer uptake was observed in patients with esophagitis, thyroiditis, arthritis, fractures, and uterine fibroids.This study provides a reference range for 18F-FAPI uptake in relevant organs and benign lesions. Benign lesion 18F-FAPI uptake may reduce 18F-FAPI PET/CT specificity.Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.