The stacking fault substructure was observed in the quenched martensite of 35CrMo, 2Cr13 and W6Mo5Cr4V2 steels by JEM-2100 transmission electron microscope. It is significant theoretically to discovery the stacking fault substructure and then to study its formation mechanism. The results show that the stacking faults in the martensite of steels are superfine with a few nanometers spacings, which are often concomitant with the high-density dislocations. It is considered that the stacking fault results from the crystal lattice misarrangement during the crystal lattice reconstruction from austenite to martensite in steels. The shear mechanism cannot explain the formation of the stacking fault.