Background. Fibrillatory Wave Amplitude (FWA) has been described as a non-invasive marker of atrial fibrillation (AF) complexity, and it predicts catheter ablation outcome. However, the actual determinants of FWA remain incompletely understood. Objective. To assess the respective implications of anatomical atrial substrate and AF spectral characteristics for FWA. Methods. Persistent AF patients undergoing radiofrequency catheter ablation were included. FWA was measured on 1-min ECG by TQ concatenation in Lead I, V1, V2, and V5 at baseline and immediately before AF termination. FWA evolution during ablation was compared to that of AF dominant frequency (DF) measured by Independent Component Analysis on 12-lead ECG. FWA was compared to the extent of endocardial low-voltage areas (LVA I < 10%; II 10–20%; III 20–30%; IV > 30%), to the surface of healthy left atrial tissue, and to P-wave amplitude in sinus rhythm. The predictive value of FWA for AF recurrence during follow-up was assessed. Results. We included 29 patients. FWA remained stable along ablation procedure with comparable values at baseline and before AF termination (Lead I p = 0.54; V1 p = 0.858; V2 p = 0.215; V5 p = 0.14), whereas DF significantly decreased (5.67 ± 0.68 vs. 4.95 ± 0.58 Hz, p < 0.001). FWA was higher in LVA-I than in LVA-II, -III, and -IV in Lead I and V5 (p = 0.02 and p = 0.01). FWA in V5 was strongly correlated with the surface of healthy left atrial tissue (R = 0.786; p < 0.001). FWA showed moderate to strong correlation to P-wave amplitude in all leads. Finally, FWA did not predict AF recurrence after a follow-up of 23.3 ± 9.8 months. Conclusions. These findings suggest that FWA is unrelated to AF complexity but is mainly determined by the amount of viable atrial myocytes. Therefore, FWA should only be referred as a marker of atrial tissue pathology