A B S T R A C TThis paper describes a method to characterize the structure of polytetrafluoroethylene (PTFE) treated gas diffusion layers (GDLs) with and without microporous layers (MPLs) using 3D X-ray micro computed tomographic (mCT) microscopy. In this work, the structure of single and dual layer GDLs is evaluated via mCT for various GDL samples (such as Toray TGP-H-060 and AvCarb EP40) loaded with different MPLs. A new method is presented for separating, or segmenting, the various phases of the GDL, i.e., void space, carbon fiber (including binder and PTFE), and MPL. Through analysis, it was found that the variation in bulk porosity and the average pore diameter of the GDLs depends highly on the GDL series manufacturing and treatment processes. Using advanced image analysis techniques, routines were developed to accurately segment the GDL fibers (including binder/PTFE) and the MPL. The percentage of the intruding MPL material into the carbon fiber paper as a function of the GDL thickness was successfully found for dual layer GDLs, with varying PTFE content and areal weight loading in the MPL. This analysis provides invaluable insight into the physical microstructure of paper-based GDLs, emphasizing the heterogeneous porosity distribution of single layer GDLs and the interaction of the MPL with the carbon fiber paper of dual layer GDLs.