The population structure of the Bacillus cereus group (52 strains of B. anthracis, B. cereus, and B. thuringiensis) was investigated by sequencing seven gene fragments (rpoB, gyrB, pycA, mdh, mbl, mutS, and plcR). Most of the strains were classifiable into two large subgroups in six housekeeping gene trees but not in the plcR tree. In addition, several consistent clusters were identified, which were unrelated to species distinction. Moreover, interrelationships among these clusters were incongruent in each gene tree. The incongruence length difference test and split decomposition analyses also showed incongruences between genes, suggesting horizontal gene transfer. The plcR gene was observed to have characteristics that differed from those of the other genes in terms of phylogenetic topology and pattern of sequence diversity. Thus, we suggest that the evolutionary history of the PlcR regulon differs from those of the other chromosomal genes and that recombination of the plcR gene may be frequent. The homogeneity of B. anthracis, which is depicted as an independent lineage in phylogenetic trees, is suggested to be of recent origin or to be due to the narrow taxonomic definition of species.The Bacillus cereus group, which is a subdivision of the genus Bacillus, includes the closely related species Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis (22). However, their pathogenic potentials and disease spectrums are quite different despite their genetic relatedness. B. cereus is an opportunistic pathogen and causes several types of infections in humans. It is frequently isolated as a contaminant of milk, cereals, and various other foods, and it produces an emetic toxin and one or several enterotoxins. On the other hand, B. thuringiensis is primarily an insect pathogen, and it produces intracellularly insecticidal crystal toxins of different specificities during sporulation or in the stationary phase, which is the only established difference between it and B. cereus (7). B. anthracis causes the potentially lethal disease anthrax, and it has been identified as a nonhemolytic, nonmotile, penicillin-sensitive, encapsulated bacterium. B. anthracis is important in that it is considered a potential biological weapon (26). The genes causing the lethal effect of anthrax are located on two large virulence plasmids, pXO1 and pXO2 (26).The genomes of these three species show high levels of similarity; for example, they share almost identical 16S ribosomal DNA sequences (2), although an association of a distinct type of 16S ribosomal DNA sequence with B. anthracis was recently reported (30). Although several phenotypes (such as capsule, lack of hemolysis, lack of motility, and susceptibility to gamma phage) and biochemical tests can differentiate B. anthracis from B. cereus and B. thuringiensis (38), species delimitation is unclear. In fact, they were suggested to be one species based on a multilocus enzyme electrophoresis (MLEE) result (14) and by the presence of an S-layer on the cell surface (24). Therefore, an ...