Oxidative damage to the genome can yield the base 8-oxo-7,8-dihydroguanine (OG). In vitro studies suggested OG would preferentially form in 5′-GG-3′ sequence contexts after exposure to reactive oxygen species. Herein, OG locations in the genome were studied by development of “OG-Seq” to sequence OG sites via next-generation sequencing at ∼0.15-kb resolution. The results of this study found ∼10 000 regions of OG enrichment in WT mouse embryonic fibroblasts and ∼18 000 regions when the OG repair glycosylase Ogg1 was knocked out. Gene promoters and UTRs harbor more OG-enriched sites than expected if the sites were randomly distributed throughout the genome and correlate with reactive 5′-GG-3′ sequences, a result supporting decades of in vitro studies. Sequencing of OG paves the way to address chemical and biological questions surrounding this modified DNA base, such as its role in disease-specific mutations and its epigenetic potential in gene regulation.