 Average nucleotide identity, ANI, is a robust and useful measure to gauge genetic relatedness between two genomes. Here, the authors develop fast ANI, a method to compute ANI using alignment-free approximate sequence mapping, and show 95% ANI is an accurate threshold for demarcating prokaryotic species by analysing about 90,000 prokaryotic genomes. This article was authored by Chirag Jain, Luis M. Rodriguez, Adam M. Felipe and others.