G-quadruplexes (G4s) are nucleic acid secondary structures that form within guanine-rich DNA or RNA sequences. G4 formation can affect chromatin architecture and gene regulation and has been associated with genomic instability, genetic diseases and cancer progression1, 2, 3, 4. Here we present a high-resolution sequencing–based method to detect G4s in the human genome. We identified 716,310 distinct G4 structures, 451,646 of which were not predicted by computational methods5, 6, 7. These included previously uncharacterized noncanonical long loop and bulged structures8, 9. We observed a high G4 density in functional regions, such as 5′ untranslated regions and splicing sites, as well as in genes previously not predicted to contain these structures (such as BRCA2). G4 formation was significantly associated with oncogenes, tumor suppressors and somatic copy number alterations related to cancer development10. The G4s identified in this study may therefore represent promising targets for cancer intervention.