Ti65 alloy has extensive potential applications in manufacturing high-temperature components in the aerospace industry. During thermomechanical processing in the α+β phase region, complicated microstructure and texture evolution occurs. In this work, isothermal hot compression experiments were conducted to comprehensively investigate the substructure and texture evolution of Ti65 alloy with bimodal microstructure via the electron backscatter diffraction (EBSD) technique, and the influence of processing parameters on the evolution of substructure and texture was systematically analyzed. The experimental results showed that the distribution of grain boundaries and the transformation of low-angle boundaries (LABs) to high-angle boundaries (HABs) exhibited sensitivity to temperature and strain rate. The fraction of sub-grain boundaries decreased, while the fraction of HABs increased with the increasing of strain rate or temperature due to the spheroidization of the lamellar α phase accompanied by the wedging of the β phase during deformation and the relative misorientation angle distribution between different α variants precipitated from β phase. Temperature rising at a high strain rate resulting from adiabatic heat promoted atomic activity, caused dislocations to be absorbed by sub-grain boundaries or HABs, and finally reduced the dislocation density in both αp and αs phases. Dynamic spheroidization was the main mechanism with increasing temperature, and coarsening of the lamellar α phase was the main mechanism with increasing strain rate. With the increase of temperature or decrease of strain rate, the texture of the αp phase became stronger and the basal and pyramidal slip could be activated. For the αs phase, spheroidization and variant selection together influence the orientation distribution. As a result, the texture evolution showed a complicated tendency.