SignificanceWe have used cryogenic electron microscopy (cryo-EM) to achieve direct, probe-free imaging of lateral domains in biomimetic lipid membranes under native conditions and to characterize differences in their structures. First, measurements of membrane thickness in laterally uniform single-component membranes show that cryo-EM is capable of subangstrom resolution of interleaflet membrane thickness. All-atom simulations are used to predict the cryo-EM appearance of submicron domains in vesicles with coexisting liquid domains and these are quantitatively validated by direct imaging of phase-separated membranes. We then extend this approach to observe nanoscopic domains in isolated cellular membranes, demonstrating direct imaging of nanodomains in biomimetic and biological membranes.