Objective: Standard stress/rest Tc-99m MIBI and T1-201 myocardial perfusion study have some limitations such as stress/rest image overlap for Tc-99m-MIBI, low energy for T1-201 and long period of study time for two separate studies. Separate acquisition rest T1-201/stress Tc-99m MIBI dual isotope study is a potentially efficient myocardial perfusion imaging protocol that combines the high resolution of Tc-99m for stress perfusion assessment and T1-201 for viability assessment. This study assessed the usefulness and diagnostic accuracy for this new approach. Methods: We tried to evaluate sensitivity and specificity of dual isotope separate acquisition protocol in 67 patients. Immediately after resting T1-201 SPECT data was acquired, dipyridamole stress Tc-99m MIBI myocardial perfusion study was performed. Visual analysis was carried out qualitatively with 0 to 3 scoring system for 17 segments of left ventricle in the reconstructed horizontal long axis and short axis slices. Results: Total study was completed within 3 hours. In angiographic correlation, dual isotope SPECT demonstrated high sensitivity(85%) and in a small group of patients, high specificity was also observed (100%). Conclusion: Combined thallium-201/stress Tc-99m MIBI SPECT displayed similiar diagnostic accuracy to protocol using stress/rest Tc-99m MIBI SPECT. This protocol was completed in shorter period than the previous protocols and therefore enhance laboratory throughput and patients convenience.