Background: Two-Dimensional Speckle Tracking Echocardiography (2D-STE) is a non-Doppler echocardiographic modality that allow quantification of myocardial deformity and its timing in 2D grey scale images, and was proved to be beneficial in assessment of dyssynchrony (STAR trial). Objective: To investigate the capability of 2D-STE for localizing Accessory Pathways (APs) in patients with Wolf-Parkinson-White (WPW) Syndrome. Patients and Methods: The prospective study included 15 patients with manifest AP indicated for invasive endocardial mapping and ablation. All patients were assessed by twelve-lead ECG, 2D-STE for the earliest activated site and invasive Electrophysiology study (EPs). Results: Out of the 15 patients with left-sided APs, 2D-STE was able to accurately localize the site of AP in 10 patents (66.6%), while electrocardiogram (ECG) was able to localize the site of AP in 12 patients (80%). Conclusion: 2D-STE has the ability to approximate the location of AP without the pitfalls of Doppler methods. However, ECG still more accurate and reproducible tool.