This study aimed to establish an optimal protocol for Tc-sestamibi parathyroid imaging for lesion localization in patients with hyperparathyroidism (HPT).We retrospectively enrolled 35 consecutive patients who underwent dual-phase (at 10 minutes and 120 minutes) Tc-sestamibi parathyroid scintigraphy with single-photon emission computed tomography (SPECT)/computed tomography (CT). Twenty seven patients had primary HPT, and 8 had secondary or tertiary HPT. Three nuclear medicine physicians independently analyzed the parathyroid images for lesion localization at 9 predefined parathyroid locations using the following 4 different image sets blinded to the clinical information:All SPECT or SPECT/CT image sets were analyzed with dual-phase planar images. The image results were compared with the histopathological results after surgery.Dual-phase SPECT/CT showed the highest positive rate of 85.7% in the patient-based analysis and 13.7% in the location-based analysis. Of 35 patients, surgical pathological results were available in 21 (16 adenomas in 16 primary HPTs and 16 hyperplasias in 5 secondary or tertiary HPTs). Dual-phase SPECT/CT showed the sensitivity values of 100% and 84.4% in the patient-based and location-based analysis, respectively, which were the highest sensitivity values among all image sets. In the primary HPT subgroup, dual-phase SPECT/CT showed the highest sensitivity value of 93.8% in the location-based analyses, whereas dual-phase SPECT, early SPECT/CT, and delayed SPECT/CT showed the sensitivity values of 62.5%, 81.3%, and 81.3%, respectively. In the secondary or tertiary HPT subgroup, dual-phase SPECT/CT also showed the highest sensitivity value of 75.0%, whereas early SPECT/CT, delayed SPECT/CT, and dual-phase SPECT showed the sensitivity values of 43.8%, 56.3%, and 68.8%, respectively.Compared with dual-phase SPECT or single-phase SPECT/CT, the dual-phase SPECT/CT imaging protocol for Tc-sestamibi scintigraphy showed the highest positive rate and sensitivity, and was optimal for parathyroid lesion localization.