A detailed study of the initiation of fatigue cracks in carbon black–filled natural rubber is conducted. Interrupted fatigue tests are performed and fatigued samples are observed with a scanning electron microscope. This procedure first enables the quantification of the morphology, spatial distribution, and evolution of crack initiation sites for different strain levels, which gives some statistical data for each strain level. It also permits analysis of the chemical nature of inclusions inducing crack initiation thanks to energy dispersive spectrometry of X-rays. It is shown that fatigue damage initially occurs generally on carbon black agglomerates or oxides such as ZnO. 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. This difference is probably because carbon black agglomerates have a stronger cohesion than ZnO inclusions and a stronger adhesion to the matrix.