In patients with secondary hyperparathyroidism, the four glands are not uniformly enlarged; therefore, preoperative localization is difficult in comparison with primary hyperparathyroidism. The aim of this study was to compare the usefulness of 99mTc-sestamibi scintigraphy versus ultrasonography in the preoperative assessment of patients with secondary hyperparathyroidism. Between October 2008 and March 2012, 25 uremic patients with secondary hyperparathyroidism underwent 99mTc-sestamibi scintigraphy and high resolution ultrasonography before total or subtotal parathyroidectomy. We measured plasma concentration of intact parathyroid hormone (PTH), calcium, phosphorus, and alkaline phosphatase (ALP) before parathyroidectomy. Sensitivity and positive predictive value (PPV), respectively, were 47.3% and 97.8% for MIBI scintigraphy, and 69.5% and 96.9% for ultrasonography. The sensitivity of combined techniques was 84.2%. There was a positive correlation between the parathyroid glands’ weight and serum calcium level, and positive MIBI scintigraphy and ultrasonography results. However, there was no correlation between the preoperative serum PTH, phosphorus, alkaline phosphatase (ALP), dialysis duration, and parathyroid glands’ weight. Ultrasonography is a reliable non-invasive localization tool. It has greater sensitivity in localizing parathyroid glands in secondary hyperparathyroidism than scintigraphy.