We report on the influence of molecular chemical structure on second harmonic activity in several homologous series of self-assembling short chain polymers containing both molecularly rigid and flexible segments and known to form supramolecular nanostructures. The second-order nonlinear optical susceptibility and physical structure of films formed by these molecules were investigated by second harmonic generation experiments and small-angle X-ray scattering. It was observed that all series of molecules formed polar films with a layered structure by self-assembly. The nonlinear susceptibilities were found to increase with the ratio of first hyperpolarizability to layer period in the self-assembled films, further suggesting spontaneous polar order in these layers of supramolecular structures.