We used reflectance difference spectroscopy (RDS) and surface differential reflectance spectroscopy (SDRS) to investigate the molecular orientation of ultrathin α-sexithiophene (α-6T) films on four passivated Si(100)-(2×1) surfaces, oxidized Si(001), water-adsorbed Si(001), hydrogen-adsorbed Si(001) and ethylene-adsorbed Si(001), in order to study the substrate dependence of the molecular orientation of isolated molecules and molecules located in islands or films. Strong in-plane anisotropy was observed on ethylene-adsorbed Si(001) even at 5nm film thickness, which was not the case for the other substrates.