The introduction of multilocus sequence typing (MLST) for the precise characterization of isolates of bacterial pathogens has had a marked impact on both routine epidemiological surveillance and microbial population biology. In both fields, a key prerequisite for exploiting this resource is the ability to discern the relatedness and patterns of evolutionary descent among isolates with similar genotypes. Traditional clustering techniques, such as dendrograms, provide a very poor representation of recent evolutionary events, as they attempt to reconstruct relationships in the absence of a realistic model of the way in which bacterial clones emerge and diversify to form clonal complexes. An increasingly popular approach, called BURST, has been used as an alternative, but present implementations are unable to cope with very large data sets and offer crude graphical outputs. Here we present a new implementation of this algorithm, eBURST, which divides an MLST data set of any size into groups of related isolates and clonal complexes, predicts the founding (ancestral) genotype of each clonal complex, and computes the bootstrap support for the assignment. The most parsimonious patterns of descent of all isolates in each clonal complex from the predicted founder(s) are then displayed. The advantages of eBURST for exploring patterns of evolutionary descent are demonstrated with a number of examples, including the simple Spain 23F -1 clonal complex of Streptococcus pneumoniae, "population snapshots" of the entire S. pneumoniae and Staphylococcus aureus MLST databases, and the more complicated clonal complexes observed for Campylobacter jejuni and Neisseria meningitidis.The ability to accurately determine the genetic relatedness of isolates of bacterial pathogens (or other disease agents) is fundamental to molecular epidemiological and evolutionary studies. In recent years, the use of nucleotide sequence variation at multiple housekeeping loci has become increasingly popular for strain characterization, as it has advantages for inferring levels of relatedness between strains and the reconstruction of evolutionary events (1, 2, 6-14, 18-23, 25, 28, 29).In many bacterial species, genetic variation at housekeeping loci accumulates as frequently or more frequently by homologous recombination (replacement of small chromosomal segments with those from related isolates) as by point mutation (15). Over the long term, recombination may prevent the true relationships between distantly related isolates of a species from being discerned. Epidemiological studies, however, are typically concerned with disease outbreaks or the spread of antibiotic-resistant or virulent strains between countries. Over these very short evolutionary timescales, of weeks to a few hundred years, recombination is unlikely to prevent the recognition of clones and clonal complexes within most bacterial populations. Thus, although the phylogenetic complexities introduced by homologous recombination may be problematic over long periods of evolutionary time (14, 15)...