Abstract: In this work, we show how field emission–scanning electron microscopy (FE-SEM) can be a useful tool for the study of self-assembled monolayer systems. We have carried out a comparative study using FE-SEM and atomic force microscopy (AFM) to assess the morphology and coverage of self-assembled monolayers (SAM) on different substrates. The results show that FE-SEM images present the same qualitative information obtained by AFM images when the SAM is deposited on a smooth substrate (e.g., mica). Further experiments with rough substrates (e.g., Al grains on glass) show that FE-SEM is capable of unambiguously identifying SAMs on any type of substrate, whereas AFM has significant difficulties in identifying SAMs on rough surfaces.