This study compares the role of microstructure on post-punching fatigue properties in three advanced high-strength steels (AHSSs) with a high-strength low-alloy (HSLA) steel commonly used in heavy-duty truck chassis. Microstructure characterization, tensile testing, high cycle fatigue (HCF) testing, fatigue crack growth rate (FCGR) testing, and neutron residual stress measurements are conducted. Punching significantly alters the microstructure, causing microstructure refinement, sub-grain formation, defect creation, tensile residual stresses, and a work-hardened shear-affected zone (SAZ) around, and a rough fracture zone, inside the punched hole. At 105 cycles, the HCF performance is primarily governed by the fatigue crack growth resistance of the as-rolled microstructure, with minimal sensitivity to punching. However, near the fatigue limit, post-punching fatigue failure is strongly related to strain localization when significant strength difference exists between microconstituents (e.g., martensite and ferrite). Strain localization also promotes sub-grain formation, reducing the local threshold stress intensity factor range (ΔKth), thus facilitating fatigue crack initiation. In microstructures with smaller strength differences (e.g., ferrite and bainite), sub-grains, together with surface roughness and residual stress, contribute significantly to the post-punching fatigue limit reduction. These findings provide insights into mechanisms of punching-induced fatigue performance degradation, offering potential strategies to optimize fatigue performance of AHSS for new applications.