Background: Recently, speckle tracking echocardiography has allowed us to easily quantify not only regional myocardial but multilayer deformation. Post-systolic shortening (PSS) is a sensitive marker of myocardial ischemia and remains even after relief from brief ischemia (ischemic memory). However, differences of ischemic memory among the layers are unclear. The aim of this study was to evaluate ischemic memory of PSS-related parameters with multilayer analysis using speckle tracking echocardiography. Methods: In 9 open-chest dogs, left ventricular short-axis images were acquired at baseline, during occlusion of the left anterior descending coronary artery (2 min), and after reperfusion (10, 20, and 30 min). Circumferential strain profiles were analyzed in three layers (endocardial, mid-wall, and epicardial). Peak systolic strain (ε S ) and strain at aortic valve closure (ε AVC ) were measured. Post-systolic index (PSI) and time to peak strain (T P ) were calculated as parameters of PSS. Results: In all three myocardial layers, ε S and ε AVC decreased during occlusion compared with baseline, but these parameters returned to the baseline levels immediately after reperfusion. In the endocardial and mid-wall layers, PSI significantly increased during occlusion, and the increase remained until 20 min after reperfusion (Figure). T P also significantly increased until 20 min after reperfusion in both layers. On the other hand, in the epicardial layer, PSI and T P did not significantly change after reperfusion because PSS did not persist. Conclusion: With multilayer analysis using speckle tracking echocardiography, ischemic memory of PSS-related parameters remained longer in the endocardial and mid-wall layers than the epicardial layer. Layer-specific analysis may be useful for assessing ischemic memory.