Aims: This study aimed to assess the efficacy of rotor and substrate guided ablation during persistent atrial fibrillation (PeAF) by measuring the organization of intracardiac electrograms (EGM) through frequency analysis. Methods: 6 patients with PeAF and no previous ablation procedure were included in the study. Ablation procedures were guided by CARTO®3 voltage maps and CardioInsight® rotors maps. A decapolar catheter positioned in the coronary sinus recorded 5 bipolar EGM at a sampling rate of 1 kHz. Frequency analysis was performed for 1-minute recordings after each region ablation. First, EGM were band-pass filtered (30-250 Hz), rectified and low-pass filtered (20 Hz). Then, power spectral density estimate was performed via Welch's method. The dominant frequency (DF) was computed and, to measure the organization of the EGM, the regularity (RI) and organization (OI) indexes were calculated. Results: A frequency gradient was observed between the proximal (≈5Hz) and distal dipoles (≈1 Hz) during PeAF but not during sinus rhythm (SR) or atrial flutter. This fact suggests atrial activity is the main contribution in the proximal dipole and ventricular activity in the distal dipole. Consequently, RI and OI increased from proximal to distal dipoles, indicating a more regular and organized component in the distal signals. However, the appearance of a predominant ventricular component in the distal EGM might be highly dependent on the electrodes position. In addition, although only 17% of patients recovered SR during ablation, OI improved between 11%-54% for 83% of patients. Rest of patients (83%) had to be cardioverted after ablation. Conclusion: Despite most patients had to be cardioverted to SR during a combined rotor and substrate ablation, OI improved after procedure in most of our patients (83%) In addition, the proximal dipole of the CS catheter is the most reliable to evaluate the OI improvement as a marker of ablation efficacy.