Ribonucleotides are now widely considered as the most frequently incorporated non-canonical nucleotides in genomic DNA. Here, we describe an approach, ribose-seq, to prepare DNA libraries for next-generation sequencing to map ribonucleotide incorporation into DNA. By specifically targeting and capturing the unique ends of alkali-derived 2′,3′-cyclic monophosphate or 2′-monophosphate from ribonucleotides embedded in DNA, ribose-seq libraries are constructed. Upon high-throughput sequencing and analysis, the distribution and identity of ribonucleotides in the genome could be determined as recently reported using yeast as a model organism1. Ribose-seq could potentially be applied to any cell type of any organism to allow profiling of ribonucleotide incorporation into genomic DNA.