We report a comparative study on the 2D self-assembly of two related ditopic benzoic acid species, which have similar shape and endgroups but different backbone symmetry. High-resolution scanning tunneling microscopy data reveal how the symmetry information of molecular building blocks is readily expressed in the resulting chiral or nonchiral supramolecular networks. The underlying square Cu(100) surface steers network orientation and accounts for carboxylate formation, resulting in an unusual intermolecular hydrogen bond motif. Our results demonstrate that symmetry and chiral resolution in 2D supramolecular assembly can be controlled via the design of functional molecules and choice of substrate.