Two new donor-acceptor (D-A) copolymers based on 1,2,5,6-naphthalenediimides (iso-NDI) and thiophene units, isoP(NDI2OD-TT) and iso-P(NDI2OD-TT), were designed from isomer chemistry and compared with the reported isomeric copolymers P(NDI2OD-T2) and P(NDI2DT-TT) to investigate the influence of isomeric structure on their optoelectronic properties. DFT calculations reveal that iso-P(NDI2OD-T2) and iso-P(NDI2OD-TT) have higher HOMO and LUMO energies and better backbone planarity relative to their isomeric polymers. IsoP(NDI2OD-T2) and iso-P(NDI2OD-TT) were synthesized by the Stille coupling polymerization and characterized by elemental analysis, H-1 NMR, GPC, UV-vis absorption, cyclic voltammetry, TGA, DSC, and organic thin film transistors (OTFTs). It was found that iso-P(NDI2OD-T2) and iso-P(NDI2OD-TT) had higher LUMO energies and broader band gaps than their isomeric ones and showed hole-dominated charge transport behavior, which is quite different from the electron-dominated charge transport feature of P(NDI2OD-T2) and P(NDI2DT-TT). In spite of the amorphous-like thin-film features, iso-P(NDI2OD-T2) exhibited high hole mobility of up to 0.3 cm(2) V-1 s(-1), and isoP(NDI2OD-TT) showed ambipolar property with hole and electron mobility of up to 0.02 and 4 x 10(-3) cm(2) s(-1), respectively.