We sought to evaluate the effects and reversibility of different locations of accessory pathways (AP) on left ventricular dyssynchrony (LVdys). The acute and chronic effects of AP were evaluated in a canine model (n = 11) and in patients with pre-excitation syndrome (n = 25). Pre-excitation was simulated in the canine model by applying VDD-type epicardial ventricular pacing near the atrioventricular (AV) groove with 50-ms AV interval after median thoracotomy, at five different sites in each animal. For the simulation of pre-excitation through the septal accessory pathway, right basal septal pacing was performed using a transvenous lead. Left ventricular dyssynchrony was measured by a two-dimensional speckle-tracking technique: before and during pacing in the canine model, and before and within 24 h after the ablation in patients with Wolff–Parkinson–White (WPW) syndrome. In the canine model, the most prominent intraventricular LVdys was observed in left lateral pre-excitation (P < 0.001). In patients with pre-excitation syndrome, LVdys was greatest in patients with left free wall accessory pathways before the ablation (P = 0.013). After catheter ablation, such a difference diminished (P = 0.619). The degree of LVdys was different according to the site of AP in both the acute model and chronic patients, and the most significant LVdys associated with pre-excitation was observed in left lateral AP. Left ventricular dyssynchrony was reversible in patients with WPW syndrome. Left ventricular dyssynchrony observed in patients with pre-excitation syndrome might be a different entity from that observed in patients with heart failure.