Polyimide (PI) films were prepared by the pulsed laser ablation, the rubbing of PI-coated substrates, the transfer of PI monolayers by vertical dipping method and the horizontal deposition of flowing water supported PI monolayers, and investigated by atomic force microscopy. A macroscopic anisotropy was measured in all these films. However, we found PI chain molecules were not uniformly oriented on a microscopic scale in the films prepared by the pulsed laser ablation and rubbing methods while a relatively perfect local and global orientation of PI chains existed in PI monolayer films.