DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes-and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage-distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.