Background: Ventricular myocardium near the accessory pathway is preexcited in patients with Wolff-Parkinson-White (WPW) syndrome. Preexcitation may result in ventricular dyssynchrony due to its abnormal ventricular activation pattern. However, the relationship between left ventricular dyssynchrony and accessory pathway location has not been elucidated. Methods: Various preexcitation including septal pathway was simulated in ten dogs with following protocol. Through median thoracotomy VDD-type epicardial ventricular pacing with 50-ms AV interval was applied near the AV groove at five different sites in each animal; right anterior, right lateral, left lateral, right posterior, and left posterior. Right basal septal pacing was performed via transjugular lead insertion. Electrocardiogram was acquired to confirm the preexcitation. Echocardiography was performed to assess standard deviation of time to peak radial strains (SRt6s) from six mid segments using 2D speckle tracking technique before and during pacing. Results: LV dyssynchrony was most prominent in left lateral preexcitation (p<0.001). Conclusion: Left lateral preexcitation could induce significant LV dyssynchrony. When considering the more prevalent LV dyssynchrony in heart failure patients, clinical significance of LV dyssynchrony and development of heart failure in WPW patients should be carefully monitored especially in patients with left lateral accessory pathway.