The gas diffusion layer (GDL) is a critical component of a proton exchange membrane fuel cell, and can play a key role in fuel cell performance. In order to design reliable and durable fuel cells, knowledge of the GDL microstructure is necessary. Currently, characterization of GDLs is generally based on porosity measurements to obtain a pore size distribution. However, the pore size distribution in GDLs may not be the only factor that affects the fuel cell performance. Additional microstructural characterization of GDLs manufactured by three different vendors (Toray, SGL, and Freudenberg) has been investigated. In addition to the pore size distribution, other statistical information of GDL microstructure including size, shape, orientation, and distribution of pores have been characterized and compared. Among these GDLs, the Freudenberg sample was found to have the smallest pore size and orientation analysis indicated that the pores were randomly distributed. Pore roundness was the lowest and pore clustering was highest in Toray sample. The effect of threshold setting on pore size data was also studied and found to have negligible influence on the calculated distributions. The microstructures of the GDLs were reconstructed in three-dimension using computer simulations and good agreement with the two-dimensional image analysis data was observed. The present work opens new opportunities for experimentalists and modelers in the area of fuel cell research to take into account the statistical characteristics of GDL microstructure.