Light-induced generation of charges into an electron acceptor–donor phase-segregated blend is studied. The blend is made of highly ordered nanoscopic crystals of 3″-methyl-4″-hexyl-2,2′:5′,2″:5″,2‴:5‴,2″″-quinquethiophene-1″,1″-dioxide embedded into a regioregular poly(3-hexylthiophene) matrix, acting as acceptor and donor materials, respectively. Kelvin probe force microscopy investigations reveal a tendency for the acceptor nanocrystals to capture the generated electrons whereas the donor matrix becomes more positively charged. The presence of particular positively charged defects, i.e., nanocrystals, is also observed within the film. The charging and discharging of both materials is studied in real time, as well as the effect of different acceptor–donor ratios. Upon prolonged thermal annealing at high temperatures the chemical structure of the blend is altered, leading to the disappearance of charge separation upon light irradiation. The obtained results allow a better understanding of the correlation between the nanoscopic structure of the photoactive material and solar-cell performance.