Due to the combination of great properties such as high specific strength, excellent fracture toughness, and good corrosion resistance, metastable β titanium alloys have been increasingly used as structural materials in the aerospace and biomedical industries. The room-temperature ductility of the metastable β titanium alloys can be improved by promoting the twinning induced plasticity (TWIP) and transformation induced plasticity (TRIP) effects. In this work, the micro- and nano-scale substructure of the deformation induced twinning in the metastable β Ti-24Nb-4Zr-8Sn and Ti-5Al-5Mo-5V-3Cr alloys was studied using scanning electron microscopy, transmission electron microscopy and aberration-corrected scanning transmission electron microscopy. High-index primary twin has been characterized in both alloys, with the nano-scale secondary twin formed in the interior and the nano-scale metastable phase layer formed at the twin boundary. The characteristics of these high-index deformation twins with hierarchical substructures will be introduced. This work is supported by the National Science Foundation, grant CMMI-2122272.