The substructures of pearlite and martensite in a water-quenched Fe-1.4C (wt %) binary alloy were investigated via optical microscopy, scanning electron microscopy and high-resolution transmission electron microscopy. In the carbide layers of the quenched pearlite lamellae, theta-Fe3C-type cementite particles and several novel carbides were observed. According to electron diffraction patterns, the crystal structure of twinned martensite could not be characterized as a body-centered tetragonal crystal structure, which is commonly assumed. Instead, the patterns suggested that the twinned martensite could be considered to have a body-centered cubic alpha-Fe {112}< 111 >-type twinning structure accompanied by a metastable omega-Fe phase (ultrafine particles) at the twinning boundaries. Furthermore, high-resolution lattice observations of twinned martensite substructures demonstrated that omega-Fe phase particles could exist independently in regions without twinning structures, indicating that they were not solely caused by overlap at twinning boundaries; thus, this prior assumption was challenged. The mechanism of the formation of new carbides in quenched pearlite and the presence of the omega-Fe phase in the regions without twinning could be reasonably explained by the autotempering or detwinning of the twinned martensite.