Introduction Electrogram (EGM) fractionation is often associated with diseased atrial tissue; however, mechanisms for fractionation occurring above an established threshold of 0.5 mV have never been characterized. We sought to investigate during sinus rhythm (SR) the mechanisms underlying bipolar EGM fractionation with high-density mapping in patients with atrial fibrillation (AF). Methods Forty-five patients undergoing AF ablation (73% paroxysmal, 27% persistent) were mapped at high density (18562 +/- 2551 points) during SR (Rhythmia). Only bipolar EGMs with voltages above 0.5 mV were considered for analysis. When fractionation (> 40 ms and >4 deflections) was detected, we classified the mechanisms as slow conduction, wave-front collision, or a pivot point. The relationship between EGM duration and amplitude, and tissue anisotropy and slow conduction, was then studied using a computational model. Results Of the 45 left atria analyzed, 133 sites of EGM fragmentation were identified with voltages above 0.5 mV. The most frequent mechanism (64%) was slow conduction (velocity 0.45 m/s +/- 0.2) with mean EGM voltage of 1.1 +/- 0.5 mV and duration of 54.9 +/- 9.4 ms. Wavefront collision was the second most frequent (19%), characterized by higher voltage (1.6 +/- 0.9 mV) and shorter duration (51.3 +/- 11.3 ms). Pivot points (9%) were associated with the highest degree of fractionation with 70.7 +/- 6.6 ms and 1.8 +/- 1 mV. In 10 sites (8%) fractionation was unexplained. The EGM duration was significantly different among the 3 mechanisms (p = .0351). Conclusion In patients with a history of AF, EGM fractionation can occur at amplitudes > 0.5 mV when in SR in areas often considered not to be diseased tissue. The main mechanism of EGM fractionation is slow conduction, followed by wavefront collision and pivot sites.