This study investigated the effect of stratified tribologically transformation structure (TTS) layers on contact fatigue crack initiation of bainitic rail steel serviced on a heavy-haul railway. Results demonstrate that the heterogeneous stratified TTS layer consists of alternating white etching layer and brown etching layer (WEL and BEL, respectively). The formation of stratified TTS is accompanied with the initiation of contact fatigue cracks, including vertical cracks, intra-layer cracks and inter-layer cracks. Unlike the current findings in conventional pearlitic rail steel, the intra-layer cracks are mostly initiated within BEL on bainitic rail steel. With help of transmission electron microcopy, nano-indentation techniques and in-situ micropillar compression tests, the initiation mechanism of contact fatigue cracks associated with stratified TTS on the bainitic rail are discussed in detail. The formation of brittle twined martensite and coarse oxidized particles is responsible for the intra-layer cracks initiated within BEL, while the inter-layer cracks nucleated from the interface are attributed to the difference in mechanical properties between different layers.