DNA-directed assembly is a well developed approach in constructing desired nano-architectures. On the other hand, E-beam lithography is widely utilized for high resolution nanoscale patterning. Recently, a new technique combining these two methods was developed to epitaxially grow DNA-mediated nanoparticle superlattices on patterned substrates. However, defects are observed in epitaxial layers which restricts this technique from building large-scale superlattices for real applications. Here we use molecular dynamics simulations to study and predict defect formation on adsorbed superlattice monolayers. We demonstrate that this epitaxial growth is energetically driven by maximizing DNA hybridization between the epitaxial layer and the substrate and that the shape anisotropy of the DNA-mediated template posts leads to structural defects. We also develop design rules to dramatically reduce defects on epitaxial layers. Ultimately, with the assist of the computational study, this technique will open the door to constructing well-ordered, three-dimensional novel nanomaterials. (C) 2016 Wiley Periodicals, Inc.