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Although Kingella kingae is the most common etiology of osteoarticular infections in young children, is a frequent cause of bacteremia in those younger than 4 years, and has been involved in clusters of invasive infections among daycare center attendees, the population structure of the species has not been systematically studied. Using multilocus sequence typing, we investigated the genetic diversity of the largest intercontinental collection of K. kingae strains to date. To facilitate typing of bacterial isolates, we developed a novel genotyping tool that targets the DNA uptake sequence (DUS). Among 324 strains isolated from asymptomatic carriers and patients from Israel, Europe, North America, and Australia with various invasive forms of the disease from 1960 to 2013, we identified 64 sequence types (STs) and 12 ST complexes (STcs). Five predominant STcs, comprising 72.2% of all strains, were distributed intercontinentally. ST-6 was the most frequent, showing a worldwide distribution, and appeared genotypically isolated by exhibiting few neighboring STs, suggesting an optimal fitness. ST-14 and ST-23 appeared to be the oldest groups of bacteria, while ST-25 probably emerged more recently from the highly evolutive ST-23. Using the DUS typing method, randomly chosen isolates were correctly classified to one of the major STcs. The comprehensive description of K. kingae evolution would help to detect new emerging clones and decipher virulence and fitness mechanisms. The rapid and reproducible DUS typing method may serve in the initial investigation of K. kingae outbreaks., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)