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Dual-phase parathyroid scan with 99mTc-sestamibi is a standardized imaging method for diagnosing parathyroid adenoma and hyperplasia. Conventional planar images using a gamma camera are performed routinely in early and delayed time points, followed by a single SPECT-CT. SPECT-CT on both early and delayed time points, although clinically useful, is not commonly performed to avoid extra radiation exposure from computed tomography (CT). This study explores the feasibility of co-registering early and delayed SPECT-CT from a single CT and evaluates the most effective combination of images for co-registration.Fourteen retrospective patients with early and delayed planar and SPECT-CT images were recruited for this validation study. Results from contemporaneous early and delayed SPECT-CT, with hardware matched registration, are considered the gold standard. Noncontemporaneous early SPECT with delayed CT and vice versa were also processed with manual alignment by an experienced and a novice operator three times each to evaluate interoperator and intraoperator variability. Maximum standardized uptake values (SUVmax) of the thyroid lobes and parathyroid adenomas were measured, and the results in terms of accuracy and precision from noncontemporaneous SPECT-CT acquisitions were evaluated.Good image quality from co-registered SPECT-CT acquired at different time points with the results showed no bias (P>0.5). The co-registration of early SPECT and delayed CT showed higher precision than the alternative combination. Overall, the experienced operator achieved better precision and intraoperator variability than the novice operator (reproducibility coefficient=33% SUV vs. reproducibility coefficient=54% SUV, P<0.001).Quantitative SUV measurement from early and delayed parathyroid SPECT-CT imaging is feasible, with the best result achieved by experienced operators using delayed CT in manual registration.