We have used scanning force microscopy and transmission electron microscopy to study the microphase separation of P(S-b-2VP) block copolymers on chemically structured substrates. Gold was patterned by microcontact printing to form regions of self-assembled alkyl monolayers terminated by −CH3 or −OH. The differences in surface and interfacial energies between the coexisting phases and the boundary surfaces strongly influence the resulting domain structure. We find that excess material accumulates only on layers formed above the H3C-terminated SAM. For this to happen, single block copolymer molecules diffuse over distances of several micrometers. TEM investigations reveal that the block copolymer is well ordered into lamellae parallel to the substrate over the HO-terminated SAM but that the block copolymer layers on the H3C-terminated SAM are frequently oriented perpendicular to the substrate. This perpendicular orientation could decrease the edge free energy of the islands that form on this layer.