In this study, the substructures and crystallographic features of as-quenched lath martensite in interstitial-free (IF) steel and low-carbon steel (0.2C steel) were investigated using focused ion beam-scanning electron microscopy serial sectioning and advanced transmission electron microscopy. A three-dimensional analysis revealed that the morphology of the so-called lath, the smallest structural unit, exhibited a wedge-shaped plate instead of the ideal lath shape. The habit plane orientations were distributed from (5 7 5)A to (1 1 1)A in the IF steel, whereas they were near to that predicted based on the phenomenological theory of martensite crystallography in the 0.2C steel. In the IF steel, well-developed dislocation cells, tangled dislocations, and curved dislocations could be observed. Conversely, straight dislocations and cementite formation were observed in the 0.2C steel. Moreover, the orientation changed significantly inside a given lath in both steels. The lath boundary comprised regularly spaced steps with (0 1 1)M atomistic terrace planes. Interfacial dislocations corresponding to the discontinuity of the (0 1 1)M lattice planes could be observed at steps, and these dislocations were introduced to compensate for the misorientation between adjacent laths. Additionally, carbon segregations were observed at the lath and block boundaries in the 0.2C steel. Our observation results on the dislocation structures, cementite formation, and segregation of carbon atoms on the lath and block boundaries indicated that the substructure changed significantly during quenching. Based on our experimental results, we discuss the origin of the substructures and crystallographic features of lath martensite.