We have investigated the crystallographic orientation relationship between ferrite and austenite and the interfacial planes between them of a low-carbon steel formed by the transformation from ferrite to austenite upon heating. Three-dimensional investigation using EBSD measurement with FIB serial sectioning technique is carried out on samples quenched from an early stage of the reverse transformation. The prior austenite orientation of martensite in the quenched microstructure is determined based on an analysis of the variants in the Kurdjumov–Sachs (K–S) relationship and the three-dimensional ferrite–austenite microstructure is successfully reconstructed. The crystallographic analysis on the three-dimensional microstructure has revealed that the nucleus of reverse-transformed austenite maintains the K–S relationship or close to it with two or three adjacent ferrite grains. The significance for reverse-transformed austenite to select these specific orientation relationships was estimated through a statistical assessment. In addition, the influence of crystallographic relationship between ferrite and austenite to nucleation and growth of reverse transformation is discussed.