Cryogenic deformation was introduced in thermomechanical processing to restrict dislocation mobility and tailor the initial dislocation distribution at the deformation stage, subsequently followed by annealing treatment to alter the grain boundary characteristic distribution with a view to the feasibility of grain boundary engineering in Incoloy 925. The microstructural evolution deformed at cryogenic (77 K) and room temperature (298 K) under various rolling reductions and annealing conditions was systematically studied to optimize the grain boundary network, which is evaluated using multi-source quantitative statistics of the twin related domains. The results showed that cryogenic deformation can adjust the dislocation accumulation mode and plastic deformation mechanism. Geometrically dislocation distributions, reconstructed by HR-EBSD device, with fishbone-like, ribbon-like or lamellar characteristics can be observed for the deformed specimens. During the subsequent annealing, the samples with 5% and 15% pre-strain are incapable of producing the ideal twin related domains, while the sample annealed at 1075°C with 10% pre-strain show the optimization of GBCD with the highest Σ3n (n = 1, 2, and 3) boundary fraction being 69.67%. Compared with the room temperature pre-deformation, cryogenic deformation can effectively reduce the critical temperature of GBE, and also leads to the Σ3n grain boundaries proportion increasing from 31.47% to 67.46% in 10%/1050°C sample. Meanwhile, with the appropriate strain, cryogenic deformation is more conducive to optimizing the GBCD than room-temperature deformation.