The long-range film structure of 15 base pair, thiol-derivatized duplexes tethered through single 3 ' and 5 ' linkages to a Au(111) surface is measured via scanning probe microscopy. Film morphology of the 3 ' linked duplexes differs from that of its 5 ' counterpart as observed by scanning probe microscopy in contact mode under buffer solution. No structural features are distinguished in a film formed with the 5 ' linked duplexes as measured with either a bare silicon nitride or chemically modified probe. However, a distinct pattern in the film structure of the 3 ' linked duplexes is measured with a chemically modified, hydrophobic tip. Small changes in linker orientation can greatly affect DNA film structure, and a hypothesis for the self-assembly mechanism of the 3 ' linked duplexes is presented. Factors such as linker placement and composition must be considered in comparing DNA microarrays formed with different tethering methods.