The invention provides purified and isolated DNA fragments from Bacillus anthracis chromosomal DNA, primer sets and probes derived therefrom, as well as kits and detection methods for B. anthracis. The methods of the invention provide for specific detection of anthrax over closely related strains of Bacillus, as well as accurate detection of low numbers of B. anthracis in an environmental sample containing large amounts of non-specific DNA. The invention is applicable to food, health care, and military applications.