AbstractUnderstanding the influence of molecular structure on charge distribution and charge separation (CS) provides essential guidance for optoelectronic materials design. Here we propose a regioisomeric strategy to tune the diverse hole‐distribution, and probe the influence on CS patterns.Para‐,meta‐andortho‐substituted benzidine‐fullerene, named1 p,1 mand1 oare designed. Following CS, hole‐delocalization occurs in1 p, while hole‐localization exists in1 mand1 o. The rates of charge separation (4.02×1011 s−1) and recombination (9.8×109 s−1) of1 pis about 20 and 12 times faster than1 mand1 o, indicating thatpara‐determined delocalization promotes ultrafast CS, whilemeta‐andortho‐generated localization contributes to long‐lived CS states. Computational analysis further implies that localization results from the destruction of electronic conjugation for1 m, and limitation of conformational relaxation for1 o. Given that the universality and simplicity of regional isomerism, this work opens up new thoughts for molecular design with tunable charge separation patterns.