Novel software allows for layer-specific evaluation of myocardial strain by speckle tracking echocardiography (2DSTE). However, the potential of layer-specific strain at rest for diagnosing coronary artery disease (CAD) in patients with suspected stable angina pectoris (SAP) remains unknown. Our objective was to evaluate the usefulness of layer-specific 2DSTE at rest for diagnosis of CAD in patients with SAP. In total, 285 patients referred with clinically suspected SAP, normal ejection fraction, and no previous cardiac history were prospectively enrolled. All patients were examined by echocardiography, including 2DSTE, exercise ECG, and coronary angiography (CAG). Layer-specific 2DSTE was performed in three apical views to provide longitudinal peak systolic strains. Stenosis >= 70% in >= 1 major coronary artery on CAG was considered as significant CAD. Of 285 patients included, 104 had significant CAD (36%). Endocardial, epicardial, and mid-myocardial GLS were all significantly impaired in CAD patients (P < 0.001). Multivariable analysis including baseline clinical parameters, conventional echocardiographic measurements, Duke score, and layer-specific strain measurements revealed epicardial [odds ratio 1.19 (P = 0.048)] and mid-myocardial [odds ratio 1.16 (P = 0.047)] global longitudinal strain (GLS) as the only independent predictors of CAD. In direct comparison, epicardial and mid-myocardial GLS had a significantly higher diagnostic performance compared to endocardial GLS (P = 0.038 and P = 0.031, respectively). In conclusion, layer-specific GLS from 2DSTE at rest was significantly impaired in patients with significant CAD. In addition, epicardial and mid-myocardial GLS were independent predictors of CAD.