Abstract: To resolve the problem of premature rolling contact fatigue (RCF) damage to wheels with non-uniform microstructure arising in their service, RCF test was conducted to investigate the influence of the wheel tread non-uniform microstructure on the RCF of wheels. The tensile properties of uniform and non-uniform microstructure were tested using in-situ tensile test bench, and the crack growth behavior was observed by scanning electron microscope. Microstructural change at RCF damaged area of specimen with non-uniform microstructure was observed by optical microscope and transmission electron microscope. The result showed that the RCF limit of non-uniform microstructure was lower than that of matrix. In addition to the normal pearlite + proeutectoid ferrite (matrix), there were a large amunt of upper bainite in the non-uniform microstructure, which interrupted the continuity and uniformity of normal matrix. RCF cracks mainly initiated and propagated at the boundary between upper bainite and matrix. Both the hardness and the elasticity of upper bainite were higher than those of the matrix while the plasticity was lower than that of the matrix. Under the same contact stress, the incongruity of the elastic-plastic deformation resulted in the stress concentration at the boundary, and thus induced and facilitated initiation and propagation of fatigue cracks in wheel steel and accelerated the occurrence of RCF damage.