Atomic substitution in an organic semiconductor alters the intermolecular and intramolecular interactions in the solid state. Thus, an understanding of such electronic interactions is crucial in designing an efficient molecule. In this study, the effect of the substitution of two hydrogen atoms in the tetracene core to sulfur in benzopyrazine-fused tetracene was investigated via theoretical methods. The reorganization energy and electronic coupling of 5,6-substituted benzopyrazine-fused tetracene and its 5,6,11,12-substituted disulfide were calculated using density functional theory. By sulfur substitution, the hole reorganization energy increases but the electron reorganization energy decreases. Meanwhile, the highest values of both hole and electron electronic couplings decrease. As a result, the calculated hole mobility decreased significantly. However, the calculated electron mobility increased slightly, indicating that the charge transport type changes from p-type to ambipolar. These results indicate that atomic substitution significantly affects the charge transport ability of organic semiconductors.