The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105 B. anthracis strains of diverse geographical origins. All B. anthracis strains produced fingerprints comprising seven to eight bands, referred to as "skeleton" bands, while one to three "diagnostic" bands differentiated between B. anthracis strains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracis strains. Anthrax is often a fatal bacterial infection that results afterBacillus anthracis spores enter a suitable host either via inhalation or ingestion or through the contamination of a wound or abrasion (8). Since death occurs within a few days after the onset of symptoms, B. anthracis presents a very serious threat if deployed as a biological weapon (13). Naturally occurring human B. anthracis infections are rare and generally result from contact with contaminated animals or their products (5).Due to the highly monomorphic nature of B. anthracis, differentiation of strains from diverse origins has proven to be very difficult. DNA fingerprinting of B. anthracis strains using ribotyping has shown some strain-to-strain variations, presumably due to changes in the organization and number of rrn loci (18). However, due to insufficient numbers of variant strains, ribotyping was found to have limited discriminatory potential.Neither arbitrarily-primed PCR fingerprints (1) nor amplified fragment length polymorphisms fingerprints (15) have been useful in discriminating between B. anthracis strains, although some genetic diversity has been observed. In this case, the diversity was due to the presence of a variable-number tandem repeat (14) sequence in the open reading frame (ORF) vrrA. Recently, seven additional loci containing these sequences were discovered in B. anthracis, leading to the identification of six genetically distinct groups of B. anthracis (16).The presence of multiple copies of highly conserved repetitive extragenic palindromic (REP) sequences in DNA (12) has been identified in a number of microorganisms (28). The technique of REP-PCR takes advantage of the fact that multiple copies of these REP sequences...