This paper studies the effect of wheel polygonalization and material properties on the fatigue failure of fastener clips in high-speed railway lines and puts forward the corresponding inhibition measures. Vibration frequencies excited by 24th-order wheel out-of-roundness (OOR) were analysed by on-site tests of the axle box vertical vibration acceleration. A finite element modelling method of polygon-shaped wheels was proposed, which was verified by comparing the field measurement with the dynamic simulation. The dynamic response of the fastener clip was simulated by using a rigid–flexible coupled model of the wheelset-track system. The effect of amplitudes as well as typical wavelengths of wheel OOR on the fracture of the clip was analysed based on the Miner fatigue damage accumulation ruler. The results show that when the wheelset ran at 237 km/h on the track, the 24th-order wheel OOR had no obvious effect on the Mises equivalent stress of the clip. When the amplitude of the 24th-order wheel OOR reached 0.225 mm and above, excited vibrations of about 1172 Hz were transmitted from the wheelset-rail system to the fastener systems, thus reducing the safe operating life of the clips. This phenomenon can be inhibited by setting a reasonable amplitude threshold for wheel re-profiling. Wheel eccentric wear (first-order OOR) and 12th-order OOR had lesser effects on the fatigue failure of the clip. The decrease of Poisson’s ratio or the increase of Young’s modulus of the material could cause the maximum Mises equivalent stress at the clip to increase, which might induce cracks, thus leading to the fracture of the clip. Therefore, during the production process, the Poisson’s ratio and Young’s modulus of fastener clips should be strictly controlled to ensure that they are within the permitted range.