It is important to characterize the pore structure of supported polycrystalline zeolite membranes but it has remained a major challenge in studying microporous inorganic membranes. This paper reports the use of positron annihilation spectroscopy including positron annihilation lifetime spectroscopy and Doppler broadening energy spectroscopy to non-destructively characterize the pore structure of four MFI zeolite membranes of different microstructures on alumina supports. Positron annihilation lifetime spectroscopy analysis reveals a bimodal pore structure consisting of intracrystalline zeolitic micropores of around 0.6 nm in diameter and irregular intercrystalline micropores of 1.4-1.8 nm in size for the four MFI zeolite membranes studied. Distributions of the micropores along the membrane thickness direction can be inferred from Doppler broadening energy spectroscopy results, illustrating development of intercrystalline gaps during the growth of the zeolite layer. The amount and size of the intercrystalline micropores of the zeolite membranes vary with the synthesis method, and are the smallest for the randomly oriented MFI zeolite membrane synthesized without template and the largest for the c-oriented MFI zeolite membrane synthesized with the template. The c-oriented membrane has an asymmetrical distribution of intercrystalline pores along the film growth direction as compared to the uniform distribution of the bimodal structure for the other three membranes. The pore structure data obtained by positron annihilation spectroscopy are consistent with the xylene isomer separation performance of these membranes. (C) 2014 Elsevier B.V. All rights reserved.