The aggregation structure of a novel polyimide (PIM-6) with six methylene flexible spacing groups in biphenyl side chain synthesized by the traditional two-step imidisation process was investigated by polarized light microscope (PLM), small angle X-ray scattering (SAXS), wide angle X-ray scattering (WAXS), and molecular simulation approach. The agreement between the experimental data and simulation result reveals that due to the predominant interchain interaction, each three backbones stack together to form a distinct lamellar cluster with side chains packed inside dispersedly. The thickness of the lamellar cluster is about 16.0A degrees, corresponding to a strong peak at 5.5 degrees in SAXS pattern. As the backbone is not perfectly parallel to each other in each lamellar cluster, the distance between each backbone ranges from 5.8 to 8.8 A degrees possibly relating to the weak peak at 9.8 degrees in WAXS pattern. Meanwhile, no birefringence or apparent phase texture has been observed by PLM indicating an amorphous nature in this film. (C) 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 2257-2261, 2010