To investigate the role of 99mTc-labeled sestamibi (99mTc-MIBI) retention level in the assessment of malignant potential of parathyroid lesions.Twenty patients with parathyroid carcinomas and forty controls with benign parathyroid lesions who underwent preoperatively 99mTc-MIBI dual-phase planar and SPECT/CT imaging were retrospectively enrolled in this study. The mean and peak of retention index (RImean and RIpeak) were measured for evaluating the retention level of 99mTc-MIBI in the parathyroid lesions. Diagnostic accuracies of RI for differentiating malignant parathyroid lesions from benign ones were assessed by receiver operating characteristic analyses (area under the curve; AUC).RIpeak (AUC =0.87, P<0.001) and RImean (AUC =0.78, P<0.001) showed significant difference between the malignant and benign lesions. The diagnostic sensitivity, specificity, positive and negative predictive values, accuracy of RIpeak were respectively 80.0%, 85.0%, 72.7%, 89.5% and 83.3% when its cutoff value was -19.03%. In addition, the level of serum parathyroid hormone (PTH) slightly correlated with RIpeak (r=0.260, P=0.044) or RImean (r=0.281, P=0.029).Parathyroid carcinomas have higher retention level of 99mTc-MIBI than benign parathyroid lesions. RIpeak may contribute to preoperative differential diagnosis of parathyroid carcinoma.