The value of gamma probes in the surgical treatment of secondary hyperparathyroidism (sHPT) was determined. The aim of this study was to enhance the rate of successful total parathyroidectomy in patients with sHPT using intraoperative gamma probe investigations. We retrospectively analyzed the clinical data of 48 sHPT patients between May 2007 and September 2011. Preoperative (99)Tc(m)- methoxyisobutyl isonitrile (MIBI) scintigraphy and high-frequency ultrasonography were used for parathyroid localization. Thirty-five patients (group I) underwent conventional neck exploration and open parathyroidectomy. Thirteen patients (group II) underwent gamma probe-guided total parathyroidectomy and parathyroid transplantation. The two groups were compared in terms of the number of parathyroid resections, operative time, and postoperative changes in the blood levels of parathyroid hormone (PTH), calcium, and phosphate. The clinical manifestations, PTH and calcium levels, age distribution, and clinical characteristics did not differ between the two groups. The accuracy of preoperative (99)Tc(m)-MIBI scintigraphy (89.74%) for the diagnosis of hyperparathyroidism did not differ from that of ultrasonography (81.25%). However, the accuracy of (99)Tc(m)-MIBI scintigraphy (66.67%) for localizing hyperfunctioning parathyroids was significantly lower than that of ultrasonography (76.86%). The operation time was significantly longer in group I ((120 ± 25) minutes) than in group II ((90 ± 30) minutes). The accuracy of parathyroid identification was significantly higher in group II (92.59%) than in group I (80.39%). On average, significantly fewer parathyroid specimens were obtained in group I (2.5 ± 0.5) than in group II (3.5 ± 0.5). Compared with group I, group II showed a significant increase (15.4%) in the number of parathyroid resections. The PTH, calcium, and phosphate levels significantly decreased postoperatively in all patients. Intraoperative gamma probe examination confirmed that the excised specimen was parathyroid tissue and improved the accuracy of parathyroid resection. The parathyroidectomy rate was increased by 15.4% due to the use of these probes. However, the probes did not detect all ectopic parathyroids, and further research is required to clarify the underlying reasons.