The pi-extended (E)-2-(2-(thiophen-2-ye-vinyl)thiophene (TVT)-based polymers are an interesting class of semiconducting polymers because of their excellent mobilities and unique film microstructures. Despite these properties, the effect of the side-chain regiochemistry of TVT skeletons on the intrinsic properties of these polymers remains unclear. To investigate this, in this study, hexyl-substituted TVT subunits with a "tail in (TI)" or "tail out (TO)" regiosymmetrical arrangement were first introduced into diketo-pyrrolopyrrole (DPP) -based copolymer main chains to afford "isomeric" polymers PI and PO, respectively. By combining optical spectroscopy, atomic force microscopy (AFM), and grazing incidence X-ray diffraction (GIXD) data, we quantitatively characterized the aggregation, crystallization, and backbone orientation of both polymer films, which were then correlated to the charge carrier mobilities. The PI film exhibited a bimodal packing motif comprising a mixture of edge-on and face-on orientations, which was beneficial for three-dimensional (3D) charge transport and resulted in a hole mobility 2-fold larger than that in the PO film (mu(h) = 1.69 cm(2) V-1 s(-1)). This comparative study substantiates the important role of the regiochemistry of TVT in developing high-performance semiconducting polymers.