Backgrounds: In patients with an accessory pathway close to the His bundle, Radiofrequency catheter ablation (RFCA) requires additional care to avoid damaging the normal conduction system. Findings suggest that the 12-lead Surface ECG during sinus rhythm is similar to the ECG of patients with anteroseptal(AS) and para-hisian accessory pathways (APs). The purpose of the study was to investigate the electrocardiographic characteristics of anteroseptal and Para-hisian accessory pathways. Methods: We retrospectively analyzed the 12-lead ECG of 10 patients with anteroseptal APs and 8 patients with para-hisian APs who underwent electrophysiologic studies. The following variables were analyzed: QRS width; R/S ratio in lead III; presence of a negative delta wave in inferior leads; precordial lead transition to R/S>1; and R-wave amplitude change of lead V1, V2 and V3 after RF ablation. Results: In the surface 12-Lead ECG, QRS width showed no statistical difference between patients with AS APs and para-hisian APs. Patients with AS APs had a higher incidence of R/S ratio>1 in lead III. All 10 patients with AS AP showed positive delta waves in the inferior leads, however 3/8 patients with para-hisian AP showed positive delta waves in lead III. The distributions of precordial lead transition to R/S>1 were no difference between AS APs (lead V2-3; n = 4, lead V3-4; n = 5, and lead V4-5; n = 1) and para-hisian APs (lead V1-2; n = 1, lead V2-3; n = 4, lead V3-4; n = 2, and lead V4-5; n = 1). Furthermore, larger amplitude of R-wave in lead V2 after RF ablation were observed in patients with para-hisian APs. Conclusions: Para-hisian APs had a higher incidence of R/S ratio<1 in lead III, negative delta waves in lead III, and larger amplitude of R-wave of lead V2 after RF ablation of the standard 12-lead ECG compared with AS APs.