We report the use of a self-assembled two-dimensional (2D) DNA nanogrid as a template to organize 5-nm gold nanoparticles (Au NPs) into periodic square lattices. Each particle sits on only a single DNA tile. The center-to-center interparticle spacing between neighboring particles is controlled to be approximately 38 nm. These evenly distributed Au NP arrangements with accurate control of interparticle spacing may find applications in nanoelectronic and nanophotonic devices.