This paper examines the single-molecule adsorption of YOYO-I-labeled lambda-DNA at compositionally patterned self-assembled monolayers (SAMs). The interactions of fluorescently labeled lambda-DNA molecule with the patterned SAMs, which are comprised of different functional groups (i.e., amine-, alcohol-, and acid-terminated thiolates), were monitored at optically transparent gold films using total internal reflection fluorescence microscopy. The role of solution pH, A-DNA concentration, and domain size was investigated. In addition to delineation of the relative adsorption strength as a function of terminal group identity (NH2 > COOH > OH), the potential importance of structural defects was also revealed. The latter result, found both at the disordered boundaries between domains and at adlayers in which structural order was affected by the length of the alkyl chain, points to the subtle but preferential adsorption of the "sticky ends" of lambda-DNA. These experiments also detected an intriguing dependence of adsorption with respect to domain size.