Objective: This prospective, small sample study aims at exploring the potential of quantitative SUV analysis of dual-phase [99mTc]Tc-MIBI SPECT/CT for preoperative localization and differentiation of hyper-functioning parathyroid lesions (PL). Materials and methods: 40 hyperparathyroid patients (M: F; 12:28, Mean Age ± SD, 40.92 ± 13.48 years) underwent SPECT/CT scintigraphy with neck and mediastinum in field of view at early (90 min) and delayed (180 min) post injection (p.i.) of [99mTc] Tc-MIBI. Images were analyzed for PL localization. Maximum and mean SUV (SUVmax; SUVmean) at 90 and 180 min p.i. for PL as well as reference thyroid tissue were measured. PL classified as adenomas, carcinoma and hyperplasia on the basis of SUV were correlated with post-surgical histopathology. Statistical analysis with paired t-test was used to measure the significance of the difference in SUV values of adenoma and hyperplasia with reference thyroid tissue. Results: SPECT/CT identified PL in 25 out of 40 patients. Using SUVmaxand SUVmean, PL were labeled as hyperplasia (02), adenomas (22), and carcinoma (01). Both SUV values showed a decline at 180 min p.i. as compared to 90 min p.i., but SUVmaxwas considered better for lesion characterization. SUVmax(g/mL) of carcinoma, adenomas, hyperplasia, and reference thyroid tissue were 16.656, 6.933 ± 2.037, 2.788 ± 0.279, and 1.117 ± 0.221 at 90 min p.i. Significant difference (P< 0.0001) of SUVmaxbetween adenomas and reference thyroid tissue but no significant difference (P= 1.000) between hyperplasia and reference thyroid tissue at both time points was found. SUVmaxat 90 min p.i. for carcinoma (16.656 g/ml) was also found higher than adenoma and hyperplasia. Parathyroid adenomas and carcinoma were confirmed on post-surgical histopathology of lesions in respective patients however two patients with hyperplasia could not undergo surgery. Conclusion: SUVmaxat 90 min p.i. of [99mTc]Tc-MIBI SPECT/CT was found to be a good quantitative parameter for PL localization and their noninvasive image-based classification.