Objective · To explore the diagnostic value of 99mTc-sestamibi (99mTc-MIBI) single photon emission computed tomography/computed tomography (SPECT/CT) and ultrasonography in hyperparathyroidism. Methods · Fifty patients with hyperparathyroidism were included. 99mTc-MIBI SPECT/CT was performed before operations in all patients, while ultrasonography was performed in 33 patients. The diagnostic efficiency was calculated for both imaging methods in comparison to pathological data. Results · Serum parathyroid hormone (PTH) levels were 352.0 (141.5–846.0) pg/mL and 1 792.0 (1 018.5–2 358.5) pg/mL, respectively, in primary hyperparathyroidism (PHPT) and secondary hyperparathyroidism (SHPT), while maximum diameters of lesion were 14.5 (9.0–20.9) mm and 10.0 (8.0–12.6) mm, respectively (both P<0.01). The accuracy of SPECT/CT were 97.7% and 62.5%, respectively (P<0.01), in PHPT and SHPT. In 33 patients, the sensitivity and accuracy of SPECT/CT were 66.3% and 74.2%, respectively, whereas the sensitivity and accuracy of ultrasonography were 45.7% and 61.4%, respectively (both P<0.05). Conclusion · Serum PTH levels were higher, while maximum diameters of lesion were longer in PHPT than that in SHPT, and the diagnostic efficiency was also higher in PHPT than that in SHPT. In the other hand, the sensitivity and accuracy of SPECT/CT were higher than that of ultrasonography.