Determining transmission electron microscope specimen thickness is an essential prerequisite for carrying out quantitative microscopy. The convergent beam electron diffraction method is highly accurate but provides information only on the small region being probed and is only applicable to crystalline phases. Thickness mapping with an energy filter is rapid, maps an entire field of view and can be applied to both crystalline and amorphous phases. However, the thickness map is defined in terms of the mean free path for energy loss (λ), which must be known in order to determine the thickness. Convergent beam electron diffraction and thickness mapping methods were used to determine λ for two materials, Si and P91 steel. These represent best- and worst-case scenario materials, respectively, for this type of investigation, owing to their radically different microstructures. The effects of collection angle and the importance of dynamical diffraction contrast are also examined. By minimizing diffraction contrast effects in thickness maps, reasonably accurate (±15%) values of λ were obtained for P91 and accuracies of ±5% were obtained for Si. The correlation between the convergent beam electron diffraction-derived thickness and the log intensity ratios from thickness maps also permits estimation of the thickness of amorphous layers on the upper and lower surfaces of transmission electron microscope specimens. These estimates were evaluated for both Si and P91 using cross-sectional transmission electron microscopy and were found to be quite accurate.