DNA polymerase fidelity is of immense biological importance due to the fundamental requirement for accurate DNA synthesis in both replicative and repair processes. Subtle hydrogen-bonding networks between DNA polymerases and their primer/template substrates are believed to have impact on DNA polymerase selectivity. We show that deleting defined interactions of that kind by rationally designed hydrophobic substitution mutations can result in a more selective enzyme. Furthermore, a single-atom replacement within the DNA substrate through chemical modification, which leads to an altered acceptor potential and steric demand of the DNA substrate, further increased the selectivity of the developed systems. Accordingly, this study about the impact of hydrophobic alterations on DNA polymerase selectivity--enzyme and substrate wise--further highlights the relevance of shape complementary and polar interactions on DNA polymerase selectivity.