Purpose This study aimed to investigate the differential findings in clinical and biochemical features, and Tc-99m sestamibi (MIBI) dual-phase parathyroid scintigraphy for malignant and benign parathyroid lesions in patients with primary hyperparathyroidism. Methods Subjects were 102 parathyroid lesions from 91 patients with primary hyperparathyroidism. Scintigraphic findings included radioactivity grade, uptake pattern, uptake contour, lesion size on early and delayed images, and degree of washout. Clinical and biochemical features were also evaluated. Histopathology confirmed the final diagnosis for all the patients. Results Final diagnoses were 94 benign parathyroid lesions and 8 parathyroid carcinomas. The patients with parathyroid carcinoma were significantly older (p=0.002) and had significantly higher serum parathyroid hormone concentrations than those with benign parathyroid lesions (p<0.001). All malignant parathyroid lesions showed intense radioactivity similar to or greater than the submandibular gland activity on delayed images (p=0.007), and little radioactivity difference between early and delayed images (p=0.012). The cancer incidence for parathyroid lesions with both intense radioactivity and no washout was 17.0% (8/47). When parathyroid lesions with all of the abovementioned findings were regarded as malignant, the cancer incidence significantly increased from 17.0% to 33.3% (8/24, p<0.001). Conclusion For Tc-99m MIBI dual-phase parathyroid scintigraphy, uptake grade on delayed images and washout were significantly useful diagnostic criteria for differentiating benign from malignant parathyroid lesions, along with age and parathyroid hormone serum concentration.