Aims: Two-dimensional speckle tracking echocardiography (2DSTE) detects early signs of left ventricular dysfunction; however, it is unknown whether layer-specific global longitudinal strain (GLS) has incremental value in diagnosis of patients with reversible ischemia assessed by single photon emission computed tomography (SPECT). Methods and Results: Eighty patients with stable angina pectoris (SAP), normal left ventricular ejection fraction (LVEF), and no history of ischemic heart disease were retrospectively identified to have been examined by 2DSTE, SPECT, and coronary angiography (CAG). Patients with a normal SPECT constituted the control group, and patients with a positive SPECT were divided into patients with or without (true- or false- positive SPECT) significant stenosis assessed by CAG. GLS was measured for two myocardial layers (endocardial and epicardial) and as well as mid-myocardial GLS. Patients with reversible ischemia had significantly lower GLS compared to the control group (GLS_{E ndocardial}: −19.0 ± 4.4% vs −21.4 ± 3.7%, P =.011; GLS_{E picardial}: −14.3 ± 2.9% vs −16.3 ± 2.9%, P =.004); GLS_{M id-myocardial}: −16.5 ± 3.6% vs −18.6 ± 3.2%, P =.006. This difference was even more evident in patients with a true-positive SPECT (GLS_{E ndocardial}: −18.0 ± 4.4% vs −21.4 ± 3.7%, P <.001; GLS_{E picardial}: −13.6 ± 3.0% vs −16.3 ± 2.9%, P <.001); GLS_{M id-myocardial}: −15.6 ± 3.6% vs −18.6 ± 3.2%, P <.001. Notably, no significant differences existed in patients with a false-positive SPECT. GLS_{E picardial} was the only independent predictor of coronary artery disease. In conclusion: In patients with SAP and preserved LVEF, layer-specific GLS at rest identifies patients with reversible ischemia. This seems to be evident only in patients with a true-positive SPECT, thus, 2DSTE at rest might improve the diagnostic accuracy of a positive SPECT.