AbstractAcetylcholinesterase (AChE) and butyrylcholinesterase (BChE) exist in a variety of oligomeric forms, each with defined cellular and subcellular distributions. Although crystal structures of AChE and BChE have been available for many years, structures of the physiologically relevant ChE tetramer were only recently solved by cryo‐electron microscopy (cryo‐EM) single‐particle analysis. Here, we briefly review how these structures contribute to our understanding of cholinesterase oligomerization, highlighting the advantages of using cryo‐EM to resolve structures of protein assemblies that cannot be expressed recombinantly. We argue that the next frontier in cholinesterase structural biology is to image membrane‐anchored ChE oligomers directly in their native environment—the cell. image