Background: Secondary hyperparathyroidism (SHPT) usually need parathyroidectomy when drug regimens fail. However, obtaining an exact preoperative map of the locations of the parathyroid glands is a challenge. The purpose of this study was to compare the diagnostic performance of ultrasonography, dual-phase 99m Tc-MIBI scintigraphy, early SPECT/CT and delayed SPECT/CT in patients with SHPT. Methods: Sixty patients with SHPT who were undergoing dialysis were evaluated preoperatively with ultrasonography, dual-phase 99m Tc-MIBI scintigraphy, early SPECT/CT and delayed SPECT/CT. Postoperative pathology served as the gold standard. The sensitivity, specificity, and accuracy rate were determined for each method. Spearman correlation analysis was used to analyze the correlation of the hyperplastic parathyroid calcification with serum alkaline phosphatase (AKP) and parathyroid hormone (PTH). Results: 229 lesions in 60 patients were pathological confirmed to be parathyroid hyperplasia with 209 lesions in typical sites, 15 lesions in upper mediastinum and 5 lesions in the thyroid. 88.33% (53/60) patients had four lesions. US, early and delayed SPECT/CT had significantly higher sensitivity and accuracy rate (P < 0.001) than did dual-phase 99m Tc-MIBI scintigraphy. Furthermore, early SPECT/CT had significantly higher sensitivity (χ2 = 17.521, P < 0.001, χ2 = 35.027, P < 0.001) and accuracy rate (χ2 = 11.076, P = 0.001, χ2 = 16.289, P < 0.001) than did US and delayed SPECT/CT. In ectopic hyperplastic parathyroid, the sensitivity of early SPECT/CT (90%) was significantly higher than US (55%) and dual-phase planar (50%) (P = 0.039 and P = 0.039). Spearman correlation results showed a significant linear association between the calcification and serum PTH (r = 0.398, P = 0.002) and AKP (r = 0.415, P = 0.002). Conclusion: The ability of early SPECT/CT to detect hyperplastic parathyroid in patients with SHPT is superior to US, 99m Tc-MIBI scintigraphy and delayed SPECT/CT, and dual-phase SPECT/CT is not essential. Calcification of parathyroid gland is related to the levels of PTH and AKP and may help provide an evidence for the identification of SHPT.