The strength-toughness combination and hardness uniformity in large cross-section 718H pre-hardened mold steel from a 20 ton ingot were investigated with three different heat treatments for industrial applications. The different microstructures, including tempered martensite, lower bainite, and retained austenite, were obtained at equivalent hardness. The microstructures were characterized by using metallographic observations, scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and electron back-scattered diffraction (EBSD). The mechanical properties were compared by tensile, Charpy U-notch impact and hardness uniformity tests at room temperature. The results showed that the test steels after normalizing-quenching-tempering (N-QT) possessed the best strength-toughness combination and hardness uniformity compared with the conventional quenched-tempered (QT) steel. In addition, the test steel after austempering-tempering (A-T) demonstrated the worse hardness uniformity and lower yield strength while possessing relatively higher elongation (17%) compared with the samples after N-QT (14.5%) treatments. The better ductility of A-T steel mainly depended on the amount and morphology of retained austenite and thermal/deformation-induced twined martensite. This work elucidates the mechanisms of microstructure evolution during heat treatments and will highly improve the strength-toughness-hardness trade-off in large cross-section steels.