The influence of the underlying substrate on the UV initiated polymerization of diacetylene lipid monolayers was investigated using absorption spectroscopy and Brewster angle microscopy (BAM). The lipid/substrate affinity was tuned through choice of lipid headgroup as well as substrate properties. Lipids with positively charged headgroups, which readily polymerized in both blue (6 °C) and red (16 °C) polymer forms at the air/water interface, failed to polymerize when transferred to glass or hydrophobic glass (headgroups facing ambient) at either temperature. BAM analysis revealed that the diacetylene film was disordered on hydrophobic glass, which likely impeded the topochemical polymerization. On glass, however, BAM showed a highly crystalline film identical to that seen at the air/water interface, suggesting that strong interactions between the positively charged lipid headgroups and the glass inhibited polymerization in this case. In agreement, when the lipid/substrate interactions were reduced, either by introducing a cadmium arachidate bilayer between the diacetylene film and the glass or by substituting mica for glass, a limited polymerization occurred, forming the red film exclusively. As a further test, monolayers of acidic diacetylene lipids were deposited on glass. In this case polymerization was possible in both blue and red forms but diminished as the transferred film was aged. These results suggest that a strong lipid/substrate affinity may impede the topochemical polymerization, possibly by restricting the mobility of the lipids. By investigating polymerization as a function of substrate and headgroup chemistry, several factors influencing the lability of diacetylene films toward topochemical polymerization are presented.