AbstractSummaryKmer-db is a new tool for estimating evolutionary relationship on the basis of k-mers extracted from genomes or sequencing reads. Thanks to an efficient data structure and parallel implementation, our software estimates distances between 40,715 pathogens in less than 4 minutes (on a modern workstation), 44 times faster than Mash, its main competitor.Availability and Implementationhttps://github.com/refresh-bio/[email protected] informationSupplementary data are available at publisher’s Web site