Combining the acceptor-donor-acceptor-type fused ring-based molecular architecture into a polymeric backbone is a promising strategy to design polymer acceptors for high-performance all-polymer solar cells (all-PSCs), and use of single isomer monomers is critical to control their physicochemical and photovoltaic properties. Here, two polymer acceptors (namely, PBI-alpha and PBI-beta) based on two different building blocks are designed and synthesized for investigating the effect of regioisomerism of the building blocks on the physicochemical and photovoltaic properties of the polymer acceptors. The subtle difference in the thienyl-fused malononitrile group of PBI-alpha and PBI-beta significantly influences their absorption spectra and electronic energy levels. PBI-alpha exhibited a lower lowest unoccupied molecular orbital level and obviously redshifted absorption spectrum compared to PBI-beta, which delivered a significantly different open-circuit voltage (0.930 +/- 0.004 vs 1.02 +/- 0.01 V) and short-circuit current density (18.7 +/- 0.3 vs 16.1 +/- 0.4 mA cm(-2)) in the PBI-alpha- and PBI-beta-based devices, respectively. Meanwhile, PBI-beta shows a more positive molecular packing effect to enhance the pi-face-on orientation backbone stacking of polymer donor PM6 than PBI-alpha, which is beneficial for efficient charge transport, leading to a higher fill factor of 0.684 in the PBI-beta-based all-PSC compared to 0.646 for the PBI-alpha-based device.