The nanoscale design and fabrication of monolayer assemblies is becoming increasingly important for research areas ranging from adhesion to chemical sensors. The formation of molecular scaffolding within a single molecular layer by linking adjacent molecules provides an important means for the nano- to microscale fabrication of such interfacial assemblies. Unfortunately, key factors in the design and fabrication of these internally linked monolayers are often overlooked due to direct analogy with the often studied n-alkyl monolayer systems. In this investigation, the impact of substrate preparation on the resulting monolayer structure is compared for n-alkyl (C18) and internally linked polydiacetylene monolayer assemblies formed on evaporated, sputtered, and colloidal gold surfaces. Polydiacetylene monolayers are fabricated by the spontaneous assembly of diacetylene-containing disulfides followed by photoinduced polymerization. The resulting polydiacetylene monolayers exhibit systematic variations in the degree of polymerization, alkyl chain crystallinity, advancing contact angle, and electron-transfer inhibition with substrate preparation. By all these measures, both the short- and long-range order of these polymerized monolayers are observed to increase substantially on evaporated gold substrates. In contrast, the n-alkyl-based monolayers formed under identical conditions show minimal structural variation. Moreover, surface pretreatment is demonstrated to have a significant impact on the long-range order for both the n-alkyl and polydiacetylene monolayers. These experimental observations implicate domain size as a significant parameter in the fabrication of polydiacetylene monolayers, while exhibiting little or no impact on the apparent structure of n-alkyl-based monolayer assemblies. Ultimately, the successful fabrication of monolayer structures containing internal molecular scaffolding is made possible by the judicious choice of substrate preparation conditions.