DNA self-assembled monolayers (SAMs) were prepared using potential-assisted deposition on clean gold single-crystal bead electrodes under a number of conditions (constant or square-wave potential perturbations in TRIS or phosphate immobilization buffers with and without Cl-). The local environment around the fluorophore-labeled DNA tethered to the electrode surface was characterized using in situ fluorescence microscopy during electrochemical measurements as a function of the underlying surface crystallography. Potential-assisted deposition from a TRIS buffer containing Cl- created DNA SAMs that were uniformly distributed on the surface with little preference to the underlying crystallography. A constant (+0.4 V/SCE) or a square-wave potential perturbation (+0.4 to -0.3 V/SCE, 50 Hz) resulted in similar DNA-modified surfaces in TRIS immobilization buffer. Deposition using a square-wave potential without Cl- resulted in lower DNA surface coverage. Despite this, the local environment around the DNA in the SAM appears to be densely packed. This implies the formation of clusters of densely packed DNA in the SAM. This effect was also demonstrated when depositing from a phosphate buffer. DNA clusters were significantly reduced when Cl- was present in the buffer. Clusters were most prevalent on the low-index plane surfaces (e.g., {111} and {100}) and less on the higher-index planes (e.g., {210} or {311}). A mechanism is proposed to rationalize the formation of DNA-clustered regions for deposition using a square-wave potential perturbation. The conditions for creating clusters of DNA in a SAM or for preventing these clusters from forming provide an approach for tailoring the surfaces used for biosensing.