Complete surgical resection of hyperfunctioning parathyroid tissue is essential for the curative treatment of hyperparathyroidism. It is very important to identify the accurate location of the affected parathyroid glands by imaging tests to successfully achieve this surgical treatment. Tc-99m labeled sestamibi is a radiopharmaceutical with high affinity for endocrinologically active parathyroid tissue, and scintigraphic evaluation using this radioactive agent is very useful in confirming the location of the affected parathyroid glands. The recent remarkable development of hand-held gamma-ray detectors makes it easy to perform radio-guided surgery, which is a combination of a preoperative intravenous injection of sestamibi and intraoperative navigation by the gamma-rays radiating from the pathological sites. This strategy can be easily applied to the resection of the ectopically located gland and is also useful to confirm the absence of endocrinologically active tissue after the operation. Additional chest SPECT imaging might be effective in the risk assessment of cardiac incidence in the perioperative period, as sestamibi was originally developed for the evaluation of myocardial perfusion. In conclusion, Tc-99m sestamibi will be more popularly applied to the treatment of hyperparathyroidism as minimally invasive surgery increases in favor.