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This work is focusing on investigating the high cycle fatigue behaviours (HCF) of Ti-6246 and TIMETAL-54M under various work conditions and improving HCF through microstructure optimization by means of thermo-mechanical processing (TMP) and shot peening (SP). TMP routes were designed to de velop different sorts of microstructures. Optical microscope (OM) and high energy synchrotron X-ray diffraction (HESXRD) were utilized to characterize the microstructures and phase constitutions, respectively. HCF of various microstructures was evaluated under rotary bending load (R-B) to clarify its microstructural dependence. The optimal microstructure was opted for surf ace strengthening by SP. To investigate the influence of loading mode, mean stress and environment, axial fatigue tests were carried out on the electro-polished (EP) optimal condition under different test parameters. The effect of SP was also determined on the peened specimens under axial load at both RT and 450 °C. Characterization of surface roughness, microhardness and residual stress depth profiles of the peened and thermo-relaxed samples was performed using profilometer, microhardness tester and hole drilling method to help interpret the role of SP playing in HCF. Positioning of the crack nucleation sites and observation of fracture characters in fatigued specimens were conducted using scanning electron microscope (SEM) to elucidate the mechanisms of crack nucleation. The results achieved by OM and HESXRD confirm the martensitic transformation in Ti-6246, which reaffirms the alloy's classification into (α + β) group. Duplex structure with 10 % primary αgrains (αp stands out for Ti-6246 with great advantage in HCF in comparison to the other microstructures and the as-received condition. The alloys exhibit high sensitivity to loading mode. Mean stress sensitivities are proven to be normal, regardless of test temperature. Increase in temperature from RT to 450 °C only results in minor decrease in HCF. The thermal stability of Ti-6246 in HCF is satisfactory. The material shows superior HCF performance in vacuum to that in the air. At RT, SP contributes a moderate improvement in HCF. As increasing temperature to 450 °C, fatigue limits are maintained. While, lifetimes at high stress amplitudes decrease due to the overwhelming detrimental effect of deteriorated surface finish after SP against the residual compressive stress. Fatigue crack nucleation (FCN) at subsurface region in EP condition is commonly observed within conventional fatigue regime (∼107 cycles), despite of load ratio. The likelihood of subsurface FCN is found to increase with the decrease of stress levels. Multi-competing crack nucleation modes are operative, strongly depending on local microstructural configuration.