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Two-dimensional speckle tracking echocardiography (2D-STE) multilayer analysis of myocardial deformation is a non-invasive method that enables discrimination of transmural differences owing to myocardial ischemia or necrosis. We wished to ascertain if multilayer longitudinal strains at rest are associated with significant (≥70 %) stenosis of the left anterior descending coronary artery (LAD) in patients with suspected non-ST-elevation acute coronary syndrome (NSTE-ACS). Our cohort comprised 113 consecutive patients with suspected NSTE-ACS and preserved ejection fraction (EF). Using coronary angiography, we diagnosed 63 patients with significant stenosis of the LAD and 50 patients without significant coronary artery disease. Echocardiography was done ≤48 h before angiography. Multilayer longitudinal strains were assessed from the endocardium, mid-myocardium and epicardium by 2D-STE. Regional longitudinal strain in LAD territory (RLS LAD ) was calculated as the mean peak systolic longitudinal strain of segments subtended by the LAD for all myocardial layers. Significant differences were observed in all strain parameters between the two groups. RLS LAD and global longitudinal strain in the endocardium showed higher accuracy than that in the mid-myocardium and epicardium, wall motion score index (WMSI), WMSI in LAD territory, and EF for detection of significant LAD stenosis (all P < 0.05), with areas under the receiver operating characteristic curve of 0.87 and 0.91, respectively. An endocardial RLS LAD cutoff of -23.52 % showed optimal sensitivity and specificity (88.9/80.0 %). In patients with suspected NSTE-ACS, multilayer longitudinal strain analysis at rest might enable prediction of significant LAD stenosis, and could help to identify patients requiring reperfusion.