To isolate and detect anthrax spores form soil in different regions of Iran in order to find the anthrax foci‚ a total of 668 environmental specimens were collected during 2003-2004. Bacterial endospores were extracted from soil specimens via flotation in distilled water, incubation at room temperature, filtration, heat shock and culture on blood agar and selective PLET media. Bacillus anthracis was identified using conventional bacteriological, biochemical and biological tests. Viable B. anthracis spores were isolated from 61 (9.1%) soil samples of the 668 collected specimens in which 21 (34.4%) isolates were encapsulated with different virulence from very high virulence to low virulence and the others (40 isolates) were unencapsulated. The isolated bacteria and their virulence were confirmed with Polymerase Chain Reaction (PCR) using B. anthracis‚ cap and pa specific primers. They also confirmed by inoculation into laboratory animals. Isolation of B. anthracis from soil in this study is reported for the first time in Iran. The encapsulated virulent bacteria were isolated from soil specimens of Isfahan, Khuzestan, West Azarbaijan, Khorasan, Charmohal Bakhtiari and Gazvin provinces. It is recommended that due to existence of highly virulent strains of B. anthracis in these regions, a review of implementation of control programs such as regular vaccination of all susceptible livestocks and surveillance of the disease in animals and human in such endemic areas is required.