Microstructure evolution during fatigue is crucial to the initiation and propagation of fatigue cracks in carbide-free bainitic steels. Utilizing quasi in-situ electron back-scattered diffraction method, the morphology of retained austenite and the accumulated plastic deformation within the bainitic laths at different fatigue stages were characterized. The observations disclosed that the retained austenite in various locations has different mechanical stability, and the dislocations in the bainitic laths will evolve to low-energy substructures continually throughout the fatigue process. Furthermore, combined with hysteresis curves analysis, a microstructural evolution model that comprehensively considered the retained austenite stability and the evolution of dislocations was proposed.