Since the introduction of technetium-99m (99mTc) sestamibi (hexakis-2-methoxyisobutyl isonitrile) as a parathyroid imaging agent in 1989, many investigators using several different imaging protocols have reported uniformly excellent results for localization of parathyroid adenomas. Exact localization of hyperplastic parathyroid glands has not met with as much success. However, the results of multiple comparative studies suggest that the diagnostic utility of sestamibi protocols equals or exceeds other noninvasive, nonscintigraphic imaging strategies, including high-resolution ultrasound, computed tomography, and magnetic resonance imaging. Two different, but not necessarily mutually exclusive imaging strategies have been used: subtraction imaging using iodine-123 (123I) or 99mTc sodium pertechnetate as the thyroid agent, and sestamibi dual-phase imaging, which takes advantage of differential washout of sestamibi from thyroid and parathyroid tissue. Sestamibi subtraction imaging has been shown to have greater sensitivity for abnormal parathyroid glands compared with thallium-201 subtraction imaging using pooled data, 87% versus 71%, respectively. Dual-phase sestamibi imaging protocols are much more variable in their conduct and have a much greater variability in sensitivity, 43% to 91%, but with a pooled sensitivity of 73%. Data suggest that dual phase techniques are at least as sensitive, and in optimized protocols, superior to, thallium-201 subtraction techniques. This superiority is attributed to the favorable washout kinetics of sestamibi and the superior imaging characteristics of the 99mTc label. Specificity and positive predictive value for both sestamibi techniques are very high, typically greater than 90% and at least equal to thallium-subtraction protocols, although specificity may be slightly lower for sestamibi subtraction techniques. Therefore, sestamibi protocols are the scintigraphic procedure of choice for parathyroid imaging. Dual-phase sestamibi protocols are more robust and lend themselves to single photon emission computer tomography (SPECT) imaging, and may be followed sequentially by subtraction techniques if results are inconclusive. Despite the excellent results of sestamibi parathyroid imaging, it is unclear whether this accuracy can compete with the even better success of an experienced surgeon in initial surgeries for hyperparathyroidism, and routine preoperative imaging before initial surgery is still controversial. However, sestamibi parathyroid imaging is an excellent addition to a correlative imaging approach in reoperations for persistent and recurrent hyperparathyroidism.