Low-energy electrons (LEEs) play an important role in nanolithography, atmospheric chemistry, and DNA radiation damage. Previously, the cleavage of specific chemical bonds triggered by LEEs has been demonstrated in a variety of small organic molecules such as halogenated benzenes and DNA nucleobases. Here we present a strategy that allows for the first time to visualize the electron-induced dissociation of single chemical bonds within complex, but well-defined self-assembled DNA nanostructures. We employ atomic force microscopy to image and quantify LEE-induced bond dissociations within specifically designed oligonucleotide targets that are attached to DNA origami templates. In this way, we use a highly selective approach to compare the efficiency of the electron-induced dissociation of a single disulfide bond with the more complex cleavage of the DNA backbone within a TT dinucleotide sequence. This novel technique enables the fast and parallel determination of DNA strand break yields with unprecedented control over the DNA’s primary and secondary structure. Thus the detailed investigation of DNA radiation damage in its most natural environment, e.g., DNA nucleosomes constituting the chromatin, now becomes feasible.