Quenching and tempering are mostly employed to tune the mechanical properties of the high-carbon steels. In the present study, transmission electron microscopy (TEM) and selected area electron diffraction (SAED) are used to examine the microstructural evolution in quenched and tempered high carbon steels. In quenched specimens, the ω-Fe(C) phase is a common substructure in twinned martensite and its diffraction spots are located at 1/3 and 2/3 (2-11)_α-Fe positions along the [011]_α-Fe zone axis (ZA). When specimens are in-situ heated in TEM, few additional diffraction spots are observed at 1/6, 3/6 and 5/6 (2-11)_α-Fe positions along the [011]_α-Fe ZA. Moreover, martensite decomposes into a lamellar structure and ω-Fe(C) phase transforms into θ-Fe₃C cementite during tempering. The TEM and electron diffraction analysis reveals that diffraction spots of θ-Fe₃C cementite phase are located at 1/6, 2/6, 3/6, 4/6 and 5/6 (22-2)_α-Fe and (2-11)_α-Fe along [112]_α-Fe and [011]_α-Fe ZAs. Furthermore, the orientation relationships between θ-Fe₃C cementite and α-Fe are indexed as: [013]_θ//[112]_α-Fe, [001]_θ//[011]_α-Fe, [-113]_θ//[111]_α-Fe and [-102]_θ//[131]_α-Fe, which are related to the transformation of ω-Fe to θ-Fe₃C cementite. The current study provides a baseline to understand the microstructural evolution in high carbon steels during heat treatment processes.