The aim of this study was to investigate the feasibility of a new technology for high-resolution and high-sensitivity SPECT imaging to differentiate the sub-epicardium and sub-endocardium using a cardiac phantom configured with normal, non-transmural and transmural myocardial perfusion defects. The phantom was filled with Tc-99m solution and was scanned using three different SPECT scanner configurations: one equipped with 19-pinhole collimator and CZT detector (Alcyone), and the other two equipped with NaI detector associated with conventional parallel hole collimators and a new spread field imaging (SFI) collimator. The Alcyone and parallel hole images were reconstructed using a commercial Ordered Subset Expectation Maximization (OSEM) software provided from GE, and the SFI images were reconstructed using the OSEM algorithm developed by Argospect. The resolution of SPECT acquired and reconstructed using the new SFI collimation and OSEM image reconstruction technology was much improved as compared to the conventional Alcyone and parallel hole images. The sub-endocardium and sub-epicardium simulated in the cardiac phantom can be clearly separated and visualized in the SPECT slices generated from the SFI technique. The simulated transmural defect was better discernable from SFI SPECT as compared to Alcyone and parallel hole SPECT. The high-sensitivity and high-resolution of the novel SFI collimation and reconstruction technology may have a great potential to identify the myocardial perfusion defects in the sub-endocardium and sub-epicardium layers from cardiac SPECT, thus improving the quantitative assessment of the transmural perfusion gradient in patients with suspected coronary artery or microvascular disease.