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Understanding the ultrafine substructure in freshly formed Fe-C martensite is the key point to reveal the real martensitic transformation mechanism. As-quenched martensite, whose transformation temperature is close to room temperature, has been investigated in detail by means of transmission electron microscopy (TEM) in this study. The observation results revealed that the freshly formed martensite after quenching is actually composed of ultrafine crystallites with a grain size of 1–2 nm. The present observation result matches well with the suggestion based on X-ray studies carried out one hundred years ago. Such nanocrystals are distributed throughout the entire martensite. The whole martensite shows a uniform contrast under both bright and dark field observation modes, irrespective of what observation directions are chosen. No defect contrast can be observed inside each nanocrystal. However, a body-centered cubic {112}<111>-type twinning relationship exists among the ultrafine α-Fe grains. Such ultrafine α-Fe grains or crystallites are the root cause of the fine microstructure formed in martensitic steels and high hardness after martensitic transformation. The formation mechanism of the ultrafine α-Fe grains in the freshly formed martensite will be discussed based on a new γ → α phase transformation mechanism.