Ti and V were bonded together and subjected to high-temperature treatment at 1000 or 1100 °C for 16 h to study the microstructural evolution and interfacial behavior of Ti–V diffusion interfaces. The samples were prepared by electro-polishing and analyzed using scanning electron microscopy, electron probe microanalysis, electron back-scattered diffraction, and nano-indentation. The results indicated that Ti–V diffusion bonding interfaces comprises a martensite Ti zone, a body-center-cubic Ti (β-Ti) zone, and a V-based alloy zone. They are divided by two composition interfaces with V contents of ~13.5% and ~46%. The original interface between the pure Ti and the V alloy substrate falls within the β-Ti zone. The observation of acicular-martensite rather than lath-martensite is due to the distortion caused by the β-to-α phase transformation in the adjacent pure Ti. The recrystallization of β-Ti is distributed along the interface direction. The hardness varies across the Ti–V interface bonding zones with the maximum value of 7.9 GPa.