A thorough analysis of the initiation stage of fatigue cracks in carbon black filled natural rubber is conducted. This study is based on interrupted tests: fatigued samples are observed with scanning electron microscope and analyzed with energy dispersive spectrometry of x-rays, in order to characterize the morphol- ogy and the chemical composition of the microstructural defects leading to the initiation of fatigue cracks. This procedure allows to quantify the spatial distribution and the evolution of crack initiation sites for differ- ent strain levels. It also reveals that inclusions such as carbon black agglomerates or ZnO are generally re- sponsible for the initiation. However, those two types of inclusions correspond to different crack initiation mechanisms and, most of the time, only the initiations on carbon black agglomerates are followed by crack propagation that leads to failure. The carbon black agglomerates appear to have a stronger cohesion than ZnO inclusions and a stronger adhesion to the matrix. Consequently, two kinds of initiation mechanisms are pro- posed considering the nature of the inclusions, their cohesion and their interface properties with the matrix.