Objective To discuss the clinical value of dual-phase 99mTc-sestamibi (99mTc-MIBI) scintigraphy and SPECT/CT in secondary hyperparathyroidism, and to investigate the possibility of SPECT/CT for the accurate localization diagnosis of secondary hyperparathyroidism preoperatively. Methods Thirty-one patients underwent parathyroid imaging with double-phase 99mTc-MIBI before surgery for hyperparathyroidism. Planar imaging was conducted in 22 patients, and planar and SPECT/CT imaging in 9 patients. The diagnosis of hyperparathyroidism was confirmed by surgical and pathological findings. Target/non-target (T/NT) ratio in delay image was compared with the volume of excised parathyroid, the intact parathyroid hormone (iPTH), and the pathology of the surgical sample. Results T/NT ratio was correlated with serum iPTH (r=0.426, P<0.05) and parathyroid volume (r=0.352, P<0.01). Hyperplasia of parathyroid gland was found in all excised samples. Planar images showed hyperparathyroidism in 73 of the 106 excised parathyroid glands, and the accuracy rate is 68.9%. In the 30 excised parathyroid glands, SPECT/CT images detected 23 hyperparathyroidism (accuracy rate 76.7%), and planar imaging found 19 hyperparathyroidism (accuracy rate 63.3%). Conclusions Dual-phase 99mTc-MIBI scintigraphy can be used to assess function and hyperplasia of parathyroid, being the most effective method for localization diagnosis of secondary hyperparathyroidism. SPECT/CT imaging is more efficient than planar imaging for the detection of parathyroid glands with hyperparathyroidism. SPECT/CT parathyroid imaging combined with CT scan will provide more information about localization of the lesions, especially for those with persistent or recurrent hyperthyroidism after parathyroidectomy.