Different austenitizing temperatures were used to obtain medium-carbon low-alloy (MCLA) martensitic steels with different lath martensite microstructures. The hierarchical microstructures of lath martensite were investigated by optical microscopy (OM), electron backscattering diffraction (EBSD), and transmission electron microscopy (TEM). The results show that with increasing the austenitizing temperature, the prior austenite grain size and block size increased, while the lath width decreased. Further, the yield strength and tensile strength increased due to the enhancement of the grain boundary strengthening. The fitting results reveal that only the relationship between lath width and strength followed the Hall–Petch formula of. Hence, we propose that lath width acts as the effective grain size (EGS) of strength in MCLA steel. In addition, the carbon content had a significant effect on the EGS of martensitic strength. In steels with lower carbon content, block size acted as the EGS, while, in steels with higher carbon content, the EGS changed to lath width. The effect of the Cottrell atmosphere around boundaries may be responsible for this change.