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The aim of this study was to evaluate the value of technetium-99m methoxyisobutylisonitrile (99mTc-MIBI) imaging and ultrasound in preoperative localization of parathyroid adenoma (PA) and parathyroid hyperplasia (PH).A retrospective study of 99mTc-MIBI double-phase scintigraphy (DPS) was performed in 187 hyperparathyroidism cases with pathologically diagnosed PA or PH. Of these patients, 167 cases underwent ultrasound, and 146 cases underwent 99mTc-MIBI single-photon emission computed tomography/computed tomography (SPECT/CT). The sensitivity and diagnostic accuracy of ultrasound, 99mTc-MIBI DPS, and SPECT/CT were compared between PA and PH. Differences in 99mTc-MIBI DPS, serum parathyroid hormone (PTH), serum calcium and phosphorus, as well as the weight and longest diameter of lesion between PA and PH were also compared.As per patient-based analysis, the sensitivity of ultrasound, 99mTc-MIBI DPS, and SPECT/CT was 90.70% (39/43), 95.56% (43/45), and 100.00% (30/30), respectively, for PA, and 93.55% (116/124), 90.85% (129/142), and 93.10% (108/116), respectively, for PH. There were no significant differences in sensitivity of these three imaging methods between PA and PH. However, per lesion-based analysis, the accuracy of ultrasound, 99mTc-MIBI DPS, and SPECT/CT in detecting PA was 78.43% (40/51), 86.79% (46/53) and 96.88% (31/32), respectively, and the accuracy of 99mTc-MIBI DPS was higher than that of ultrasound (χ2=6.507, P=0.011), and for PH, it was 49.69% (160/322), 40.71% (171/420), and 43.80% (152/347), respectively. The accuracy of ultrasound was higher than that of 99mTc-MIBI DPS (χ2=5.940, P=0.015). The accuracy of a combination of all three examinations of ultrasound+99mTc-MIBI DPS, ultrasound+99mTc-MIBI SPECT/CT, 99mTc-MIBI DPS+SPECT/CT, and ultrasound+99mTc-MIBI DPS+99mTc-MIBI SPECT/CT was 51.51% (154/299), 53.85% (161/299), 50.17% (150/299), and 54.18% (162/299), respectively, which was higher than that of ultrasound (χ2=5.273, P=0.022; χ2=8.226, P=0.004; χ2=3.880, P=0.049; χ2=8.702, P=0.003, respectively). Serum levels of PTH and phosphorus were lower in patients with PA than in patients with PH (P<0.001), and serum calcium level, the weight, and the longest diameter of lesion and early uptake rate of 99mTc-MIBI DPS were higher in patients with PA than in patients with PH (P<0.01). Serum PTH level is often less than 1000 pg/ml in PA, but usually more than 1000 pg/ml in PH.Ultrasound, 99mTc-MIBI DPS, and SPECT/CT all have a higher value in the diagnosis of PA than PH. 99mTc-MIBI SPECT/CT should be optimal for detecting PA, and early SPECT/CT scan might be better than delayed scan. Compared with 99mTc-MIBI DPS and SPECT/CT, ultrasound has a slight advantage in localization of PH lesions. The combination of ultrasound and 99mTc-MIBI DPS or SPECT/CT imaging could improve the accuracy in localization of PH lesions and should be considered as the first-line method for detecting PH.