Understanding 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-and orthosubstituted benzidine-fullerene, named 1 p, 1 m and 1 o are designed. Following CS, hole-delocalization occurs in 1 p, while hole-localization exists in 1 m and 1 o. The rates of charge separation (4.02 × 10 11 s À 1 ) and recombination (9.8 × 10 9 s À 1 ) of 1 p is about 20 and 12 times faster than 1 m and 1 o, indicating that para-determined delocalization promotes ultrafast CS, while meta-and ortho-generated localization contributes to long-lived CS states. Computational analysis further implies that localization results from the destruction of electronic conjugation for 1 m, and limitation of conformational relaxation for 1 o. Given that the universality and simplicity of regional isomerism, this work opens up new thoughts for molecular design with tunable charge separation patterns.