Purpose: Although automatic quantification software of myocardial perfusion SPECT provides highly objective and reproducible quantitative measurements, there is still some limitation in the direct use of quantitative measurements. In this study we derived parameters using normal variation of perfusion measurements, and tried to test the usefulness of these parameters. Materials and Methods: In order to calculate normal variation of perfusion measurements on myocardial perfusion SPECT, 55 patients (M:F=28:27) of low-likelihood for coronary artery disease were enrolled and 201Tl rest / 99mTc-MIBI stress SPECT studies were performed. Using 20-segment model, mean (m) and standard deviation (SD) of perfusion were calculated in each segment. As a myocardial viability assessment group, another 48 patients with known coronary artery disease, who underwent coronary artery bypass graft surgery (CABG) were enrolled. 201Tl rest / 99mTc-MIBI stress / 201Tl 24-hr delayed SPECT was performed before CABG and SPECT was followed up 3 months after CABG. From the preoperative 24-hr delayed SPECT, Qdelay (perfusion measurement), Δdelay (Qdelay–m) and Zdelay ((Qdelay–m)/SD) were defined and diagnostic performances of them for myocardial viability were evaluated using area under curve (AUC) on receiver operating characteristic (ROC) curve analysis. Results: Segmental perfusion measurements showed considerable normal variations among segments. In men, the lowest segmental perfusion measurement was 51.8±6.5 and the highest segmental perfusion was 87.0±5.9, and they are 58.7±8.1 and 87.3±6.0, respectively in women. In the viability assessment, Qdelay showed AUC of 0.633, while those for Δdelay and Zdelay were 0.735 and 0.716, respectively. The AUCs of Δdelay and Zdelay were significantly higher than that of Qdelay (p=0.001 and 0.018, respectively). The diagnostic performance of Δdelay, which showed highest AUC, was 85% of sensitivity and 53% of specificity at the optimal cutoff of -24.7. Conclusion: On automatic quantification of myocardial perfusion SPECT, the normal variation of perfusion measurements were considerable among segments. In the viability assessment, the parameters considering normal variation showed better diagnostic performance than the direct perfusion measurement. This study suggests that consideration of normal variation is important in the analysis of measurements on quantitative myocardial perfusion SPECT.