SummaryChemical modifications to nucleobases have a great influence on various cellular processes, by making gene regulation more complex, thus indicating their profound impact on aspects of heredity, growth, and disease. Here, we provide the first genome-wide map of 5-formyluracil (5fU) in living tissues and evaluate the potential roles for 5fU in genomics. We show that an azido derivative of (2-benzimidazolyl)acetonitrile has high selectivity for enriching 5fU-containing genomic DNA. The results have demonstrated the feasibility of using this method to determine the genome-wide distribution of 5fU. Intriguingly, most 5fU sites were found in intergenic regions and introns. Also, distribution of 5fU in human thyroid carcinoma tissues is positively correlated with binding sites of POLR2A protein, which indicates that 5fU may distributed around POLR2A-binding sites.