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Abstract: The microstructures of 25CrMo48V martensitic steel quenched at the different temperatures of 800–1200°C and tempered at 650°C were characterized by optical microscope (OM), field emission scanning electron microscopy (FESEM), electron backscattering diffraction (EBSD) and transmission electron microscopy (TEM). The tensile and impact properties were evaluated from the as-quenched and tempered specimens. The results show that the refinement of the prior austenite grains due to the decrease of quenching temperature could induce the refinement of packets and blocks, while the lath width remained stable. The yield strength and the 50% fraction appearance transition temperature (50% FATT) varied linearly with the reciprocal square root of the prior austenite grain size, packet size and block width. Their relationships followed the classical formula of Hall–Petch and , respectively. The prior austenite grain size has a remarkable effect on the strength and toughness, while the block is the minimum structure unit controlling strength and toughness. [Copyright &y& Elsevier]