The fabrication of well-defined interfacial structures has been recently facilitated by incorporating diacetylenic structures within spontaneously formed monolayer assemblies, Photoinduced polymerization within these single molecular layers provides a robust system for interfacial design that can be controlled using phototemplating. In this paper, we examine the control of molecular structure within this single molecular layer by introducing internal and external perturbations to the monolayer fabrication process. Internal steric perturbations are assessed by incorporation of an ester moiety below the polymer backbone. Direct comparison of monolayer structures formed using analogous precursors with and without the ester functionality indicates significant disruption in the alkyl crystallinity in the region below the polymer backbone. Despite the considerable constraints on the structural order required for successful polymerization, this steric disruption does not appear to significantly limit the polymerization process, The effect of external perturbations on monolayer polymerization is evaluated using an atomically rough substrate. Under these conditions, selective polymerization appears to occur in the interstitial regions between substrate features, Such localized polymerization has clear implications for nanoscale templating in the fabrication of well-defined interfacial structures. Further control over the interfacial chemistry is facilitated by the addition of a secondary adsorbate after monolayer polymerization. This combination of internal and external control over monolayer fabrication makes possible both the long-and short-range control of interfacial chemistry that is important for applications ranging from sensor design to adhesion studies.