Two-dimensional (2D) assemblies of alkoxy-substituted oligo(phenylene-ethynylene)s bearing different substituents adsorbed on highly oriented pyrolytic graphite (HOPG) were studied by using scanning tunneling microscopy. It was found that the introduction of different endgroups or a biethynylene linkage into oligo(phenylene-ethynylene)s can significantly change their 2D ordering on HOPG. The carboxylic endgroups can direct the conjugated oligomers to form ordered lines through intermolecular hydrogen bonding. The possibility of controlling the 2D assemblies of conjugated molecules is of importance in designing organic optoelectronic devices.