Mo/Si soft x‐ray multilayer films have been imaged in cross section by high resolution electron microscopy and the dependence of important multilayer parameters on imaging conditions has been investigated. Multiple measurements of the widths of the Mo crystalline layers were made from images recorded as part of a through‐focal series. The layer thicknesses were found to be insensitive to the focus setting near the Scherzer defocus value (+160 A˚ to −800 A˚). At larger defocus conditions, however, additional Mo fringes were visible so that the Mo crystalline region seemed to become thicker at the expense of the pure Si and interlayer regions. Micrographs recorded with the ML slightly tilted (∼1–2°) from the edge‐on orientation suggested thicker Mo and thinner Si and interlayer regions but the apparent bilayer periodicity did not change measurably. The possibility of changes in multilayer structure induced during cross‐sectional specimen preparation was also shown. Finally, the results of the image analysis are compared with those obtained from small and large angle x‐ray scattering experiments from the same sample.