Outbreaks of Bacillus anthracis in animals are repeatedly reported in the Islamic Republic of Iran. In this study soil samples were analysed from endemic regions of the country, and B. anthracis isolates were identified by classical bacteriological and biochemical methods. A multiplex polymerase chain reaction (PCR) assay was also developed as an alternative for identification of isolates, and was shown to be a rapid, sensitive and specific diagnostic assay. The results confirmed that 25 samples contained B. anthracis, of which 9 were virulent for mice and guinea pigs. This study suggests that multiplex PCR can be used as a reliable alternative for the detection of B. anthracis spores.