Twin formation in hematite during dehydration was investigated using X-ray diffraction, electron diffraction, and high-resolution transmission electron microscopy (TEM). When synthetic goethite was heated at different temperatures between 100 and 800 °C, a phase transformation occurred at temperatures above 250 °C. The electron diffraction patterns showed that the single-crystalline goethite with a growth direction of [001]G was transformed into hematite with a growth direction of [100]H. Two non-equivalent structures emerged in hematite after dehydration, with twin boundaries at the interface between the two variants. As the temperature was increased, crystal growth occurred. At 800 °C, the majority of the twin boundaries disappeared; however, some hematite particles remained in the twinned variant. The electron diffraction patterns and high-resolution TEM observations indicated that the twin boundaries consisted of crystallographically equivalent prismatic (100) (010), and (1 $$\bar{1}$$ 0) planes. According to the total energy calculations based on spin-polarized density functional theory, the twin boundary of prismatic (100) screw had small interfacial energy (0.24 J/m2). Owing to this low interfacial energy, the prismatic (100) screw interface remained after higher-temperature treatment at 800 °C.