A RSI-PCR assay was developed for the detection of a Bacillus anthracis-specific nonsense mutation in the plcR gene. The assay specificity was tested using 170 Bacillus spp. strains including 47 strains of B. anthracis. The plcR RSI-PCR distinguished Bacillus cereus group strains closely related to B. anthracis from the anthrax agent. The assay was found to be a robust, simple and cost effective tool for B. anthracis identification. In contrast to previously developed real time PCR-based methods, the RSI-PCR needs basic molecular biology equipment only, and thus may be easily introduced in developing countries, where anthrax is endemic.