Aims: To identify a chromosomal marker with signature nucleotides specific for Bacillus anthracis . Methods and Results: Repetitive element polymorphism-polymerase chain reaction with BOX-A1R primer was used to discriminate 52 strains of all six species of the 'B. cereus group'. A B. anthracis signature fragment, named AC-390, was cloned and sequenced. The deduced amino acid sequence was homologous to that of YwfK of B. subtilis . Using two internal primers, the AC-390 fragment was sequenced from two other B. anthracis strains as well as from strains of B. cereus and B.thuringiensis which have an AC-390 fragment homologous to that of B. anthracis as shown by Southern hybridization experiments. Conclusions: Two new signature sequences specific for B. anthracis were identified on a chromosomal fragment homologous to YwfK, a transcriptional regulator of B. subtilis . Significance and Impact of the Study: These results show a new chromosomal DNA trait useful for distinguishing B. anthracis from the related species of the B. cereus group, regardless of the presence of the virulence plasmids pXO1 and pXO2.