One of the major challenges to the fabrication of functionalized templates using self-assembled monolayers (SAMs) is the characterization of nanoscale defects, particularly SAM domain boundaries (DBs). In this study, an etchant was used to chemically amplify the DBs in a SAM by forming microscale pits in the underlying SiO2 layer. This approach revealed that the naturally occurring DBs acted as structural defects in the SAMs. The DB structures were characterized by systematically varying the octadecyltrichlorosilane (ODTS) monolayer domain size from the nanoscale to the microscale by varying the preparation temperature. These approaches showed that the SAM DBs, which were visualized as having round- and line-shaped nanoscale structures, provided potentially chemical and mechanical surface defect sites. Our principal findings open up exciting new possibilities for understanding the structural defects in SAMs on the molecular level and suggest an approach for optimizing the conditions used to generate defect-free SAM templates.