As one of the most effective molecular design strategies in organic electronics, heteroatom substitution was employed for the first time to study the acceptor variation effects on the optical, electrochemical, molecular assembling, and charge-transport properties of novel semiconducting polymers containing benzobisthiadiazole (BBT)-related heterocycles, namely, poly(dithiazolfluorene-alt-thiadiazolobenzotriazole) (PSN), poly(dithiazolfluorene-alt-selenadiazolobenzotriazole) (PSeN), and poly(dithiazolfluorene-alt-selenadiazolobenzothiadiazole) (PSeS). The effect of the heteroatom substitution was clearly shown in the UV-vis-NIR absorption spectra in which the substitution of the sulfur (S) and/or nitrogen (N) atoms in PSN with the selenium (Se) and sulfur (S) atoms led to a red-Shift in the absorption profile. In addition, the energy levels of these polymers, determined from cyclic voltammetry (CV) measurements and density functional theory (DFT) calculations, also varied due to the hetroatom substitution effect. Accordingly,thin film transistors (TFTs) based on these polymers showed different charge transport properties. For example, PSN displayed p-type unipolar performances with a high hole mobility up to 0.65 cm(2) V-1 s(-1). In contrast, PSeS showed n-type dominant charge transport properties with an electron mobility up to 0.087 cm(2) V-1 s(-1). Intriguingly, PSeN exhibited ambipolar charge transport properties with balanced mu(h) and mu(e) values. These different charge polarities in the TFTs were correlated to the energy levels, pi-pi stacking distances, and polymer crystallinities evaluated by their grazing-incidence wide-angle X-ray scattering (GIWAXS) patterns and atomic force microscopy (AFM) images. We believe that this simple and effective approach will guide the way to developing high-performance ambipolar and/or n-channel semiconducting polymers for TFTs.