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Accurate preoperative localization of parathyroid lesion(s) is crucial for successful surgical management of primary hyperparathyroidism (PHPT). This study was conducted to compare the effectiveness of ultrasonography (USG) of the neck, 99mTc-sestamibi (MIBI) imaging with or without SPECT/CT, and 18F-fluorocholine (FCH) PET/CT imaging in the preoperative localization of parathyroid lesions in patients with PHPT.Fifty-four consecutive patients with PHPT were included in this prospective study who underwent preoperative localization of the parathyroid lesion(s) using 3 diagnostic modalities followed by surgery. The sensitivity, positive predictive value, and accuracy of the 3 imaging procedures to accurately detect abnormal parathyroid glands were determined using histopathology as criterion standard with postoperative biochemical response confirmation.18F-fluorocholine PET/CT detected 52 of 54 patients and 52 of 56 lesions with histopathologically proven parathyroid adenomas on patient-based and lesion-based analysis, respectively. Preoperative USG, MIBI, and FCH PET/CT localized abnormal parathyroid gland(s) in 39 (72.2%), 43 (79.6%), and 54 (100%) patients, respectively. The sensitivity and positive predictive value were 69.3% and 87.1% for USG, 80.7% and 97.6% for MIBI, and 100% and 96.3% for FCH PET/CT. The accuracy was 62.9%, 79.6%, and 96.3% for USG, MIBI, and FCH PET/CT, respectively, in patient-wise analysis. In 6 patients with ectopic lesions, FCH PET/CT demonstrated higher sensitivity and accuracy than MIBI and USG (100% vs 66.6% and 16.7%, respectively).Among the 3 imaging techniques tested simultaneously, FCH PET/CT was superior for accurate preoperative localization of parathyroid adenomas, especially for ectopic or small parathyroid lesions.