We have investigated the influence of micrometer- and sub-micrometer-scale surface heterogeneities in patterned octadecyltrichlorosilane (OTS) films on human serum albumin (HSA) adsorption and its spatial distribution. 5-μm-wide OTS patterns were created on glass substrates by micro-contact printing and in some instances subsequent annealing was used to alter OTS molecule distribution within the patterns. Scanning force microscopy (SFM), advancing water contact angles and water vapor condensation figures were used to characterize the OTS films and to assess the nature of the heterogeneities within the various surface areas. High-resolution fluorescence microscopy was used to record images of fluorescently labeled albumin on OTS patterned films and fluorescence intensity was quantified and converted into the adsorbed amount. Adsorbed albumin was also characterized through SFM measurements. Combined SFM topography and lateral force microscopy (LFM) imaging revealed that micro-contact printing of OTS onto glass both replicated the stamp pattern and created small islands within the non-stamped regions between the patterns. The OTS coverage within stamped regions was not fully continuous but improved with subsequent annealing. Annealing also resulted in OTS island growth within the non-stamped regions and decreased average wettability on both the stamped and non-stamped areas. The extent of albumin adsorption was not proportional to OTS coverage, but correlated with the sub-μm distribution of OTS chains. We inferred that the surface distribution of ligands such as OTS on a sub-μm length scale determines the nature of albumin adsorption and its kinetics.