The effect of heat input on the microstructure and toughness of simulated subregions was investigated by welding thermal simulations and Charpy impact tests. The results indicate that the microstructures of the simulated coarse and fine grain heat affected zone (CGHAZ and FGHAZ) gradually changed from lath martensite to a mixture of lath martensite/lath bainite and finally to granular bainite with the increase in heat input. The microstructure of the simulated intercritical heat affected zone (ICHAZ) was mainly composed of granular bainite and blocky martensite regardless of heat input. When the heat input increased, the toughness of the simulated CGHAZ and ICHAZ continuously decreased. Nevertheless, the simulated FGHAZ still displayed good toughness (53.16 J) due to its fine structure. The occurrence of martensite-austenite (M-A) constituents was the main reason for the decrease in crack initiation energy of the simulated CGHAZ and ICHAZ at high values of heat input, and the toughness deteriorated as the size of M-A constituents increased. It should be noted that high-misorientation packet and block boundaries can effectively deviate or arrest the propagation of microcracks. When the heat input was in the approximate range of 8.63-14.95 kJ cm⁻¹, all of the simulated subregions exhibited good toughness.