ABSTRACT Comparative sequence analysis was performed upon Bacillus anthracis and its closest relatives, B. cereus and B. thuringiensis . Portions of rpoB DNA from 10 strains of B. anthracis , 16 of B. cereus , 10 of B. thuringiensis , 1 of B. mycoides , and 1 of B. megaterium were amplified and sequenced. The determined rpoB sequences (318 bp) of the 10 B. anthracis strains, including five Korean isolates, were identical to those of Ames, Florida, Kruger B, and Western NA strains. Strains of the “ B. cereus group” were separated into two subgroups, in which the B. anthracis strains formed a separate clade in the phylogenetic tree. However, B. cereus and B. thuringiensis could not be differentiated. Sequence analysis confirmed the five Korean isolates as B. anthracis . Based on the rpoB sequences determined in the present study, multiplex PCR generating either B. anthracis -specific amplicons (359 and 208 bp) or cap DNA (291 bp) in a virulence plasmid could be used for the rapid differential detection and identification of virulent B. anthracis .