Objective To investigate the association between imaging findings and histopathological characteristics of parathyroid glands in patients with secondary hyperparathyroidism (SHPT). Methods Seventy-four glands from 21 patients with SHPT who underwent parathyroidectomy were evaluated for their pathological characteristics. The detection rates of parathyroid glands using ultrasound (US) and 99Tc-MIBI-SPECT/CT (MIBI) were compared. Glands were classified as either US-positive or US-negative, and MIBI-positive or MIBI-negative. Morphological and pathological differences between the positive and negative groups were systematically analysed. Results The detection rates for parathyroid glands were 71% with US, 65% with MIBI, and 82% when combining both methods. US and MIBI showed similar localization accuracy in SHPT (P = .38). MIBI-positive glands had significantly larger oxyphil nodules compared with MIBI-negative glands (area: 10.92 mm(2) vs 3.09 mm(2), P < .01; area proportion: 61% vs 30%, P = .002), while no significant differences were found in chief nodules. The US-positive group had fewer and smaller chief nodules (number: 2 vs 9, P = .005; area: 1.53 mm(2) vs 11.08 mm(2), P = .033) and a higher percentage of oxyphil nodules (74% vs 33%, P = .003) compared with the US-negative group. Thirteen glands undetected by both US and MIBI had smaller oxyphil nodule areas (3.59 vs 13.24 mm(2)) and lower oxyphil nodule area percentages (25% vs 68%). These pathological features, including adipose infiltration, intra-gland haemorrhage, cyst formation, and calcification, showed no correlation with the gland's imaging results. Conclusion US and MIBI had similar value in preoperative localization of SHPT. Parathyroid glands with more and larger oxyphil nodules were more likely to be detected by both MIBI and US.