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 ortho-substituted benzidine-fullerene, named 1p, 1m and 1o are designed. Following CS, hole-delocalization occurs in 1p, while hole-localization exists in 1m and 1o. The rates of charge separation (4.02×1011 s-1) and recombination (9.8×109 s-1) of 1p is about 20 and 12 times faster than 1m and 1o, 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 1m, and limitation of conformational relaxation for 1o. Given that the universality and simplicity of regional isomerism, this work opens up new thoughts for molecular design with tunable charge separation patterns.