The influence of membrane microstructure on the transport properties of ZSM-5 membranes was investigated. Two zeolite membranes with (101)- and (002)-orientations were grown layer-by-layer onto seeded alumina support. The membrane morphology was kept constant as well as the shape of the individual crystal grains that made up the polycrystalline zeolite membrane layer. The membrane microstructure were characterized and quantified using six micro structural parameters that include membrane thickness (tau), grain size (d), grain morphology (M), zeolite population (N), crystal intergrowth (I-c) and film orientation. Eight different gases including He, H-2, N-2, Ar, CH4, n-C4H10, i-C4H10 and SF6 were used as molecular probes to investigate the transport processes through the membrane of different thicknesses. By maintaining a comparable non-zeolite flow, it was demonstrated that the (101)- and (002)-oriented ZSM-5 membranes have comparable transport resistance. Also, the results of the multi-thickness comparison using the different sized molecular probes indicate a strong similarity in the transport mechanism and diffusion pathway through these two membranes. The experiment suggests that the grain boundary is the main non-zeolite diffusion pathway in the membrane and their elimination through grain growth can result in better membrane performance.