Deep cryogenic treatment of tool steels, incorporated in conventional hardening and tempering, has been a topic of intensive research in recent years. Yet, the governing microstructural mechanisms involved in the deep cryogenic treatment of high-alloyed tool steels are still controversial. Thus, an in situ X-ray diffraction study is conducted on three tool steels X38CrMoV5-3, X153CrMoV12, and ~X190CrVMo20-4 to shed light on microstructural evolution during cryogenic treatment and subsequent tempering. For these high-alloyed tool steels, the transformation of retained austenite into martensite is detected during the cooling phase of the cryogenic treatment. A change in tetragonality of martensite occurs mainly in the heating phase of the subsequent tempering process, which indicates the diffusion of carbon and carbide precipitation from the martensite. The microstructure evolution of the tool steels after hardening, cryogenic treatment, and tempering is further examined by scanning electron microscopy. © 2021 The Authors. Steel Research International published by Wiley-VCH GmbH