Anthrax is an infectious disease caused by Bacillus anthracis, that occurs most frequently in herbivores and less often in omnivores, carnivores and humans. The aim of the study was application of Real-time PCR based on SYBR Green I intercalating dye for detection of B. anthracis in biological samples, like blood and liver. The experiment involved four vaccinal and two field strains of B. anthracis and also other Bacillus species. For evaluation of the specificity of tests six strains of other species of bacteria were used additionally. DNA was isolated using kits Genomic Blood Mini or Genomic Mini, A&A Biotechnology. The QuantiTect SYBR Green PCR Kit, Qiagen was used in the studies. Two genes responsible for virulence – pag and capC, located on plasmids pXO1 and pXO2, were used as markers to detect B. anthracis in contaminated biological samples in Real-time PCR. A test for presence of chromosomal sequence rpoB was also performed. On the basis of observation of amplification curves in Real-time PCR, examined genes were detected in blood and liver samples experimentally contaminated with six strains of B. anthracis. The specificity of amplification was confirmed by determination of the melting temperature of PCR products. Tests were significantly sensitive and highly specific. The applied SYBR Green I Real-time PCRs allowed detection of the virulence markers and the specific chromosomal sequence in biological samples contaminated with B. anthracis