An understanding of protein conformational ensembles is essential for revealing the underlying mechanisms of interpeptide recognition and association. However, experimentally resolving multiple simultaneously existing conformational substates remains challenging. Here, we report the use of scanning tunneling microscopy (STM) to analyze the conformational substate ensembles of β sheet peptides with a submolecular resolution (in-plane <2.6 Å). We observed ensembles of more than 10 conformational substates (with free energy fluctuations between several k _B T s) in peptide homoassemblies of keratin (KRT) and amyloidal peptides (−5Aβ42 and TDP-43 341–357). Furthermore, STM reveals a change in the conformational ensemble of peptide mutants, which is correlated with the macroscopic properties of peptide assemblies. Our results demonstrate that the STM-based single-molecule imaging can capture a thorough picture of the conformational substates with which to build an energetic landscape of interconformational interactions and can rapidly screen conformational ensembles, which can complement conventional characterization techniques.