AbstractG-quadruplexes (G4s) are four-stranded DNA secondary structures that form in guanine-rich regions of the genome. G4s have important roles in transcription and replication and have been implicated in genome instability and cancer. Thus far most work has profiled the G4 landscape in an ensemble of cell populations, therefore it is critical to explore the structure–function relationship of G4s in individual cells to enable detailed mechanistic insights into G4 function. With standard ChIP-seq methods it has not been possible to determine if G4 formation at a given genomic locus is variable between individual cells across a population. For the first time, we demonstrate the mapping of a DNA secondary structure at single-cell resolution. We have adapted single-nuclei (sn) CUT&Tag to allow the detection of G4s in single cells of human cancer cell lines. With snG4-CUT&Tag, we can distinguish cellular identity from a mixed cell-type population solely based on G4 features within individual cells. Our methodology now enables genomic investigations on cell-to-cell variation of a DNA secondary structure that were previously not possible.