Updated project metadata. Anthrax is a zoonotic infection caused by the bacterium Bacillus anthracis (BA), a gram-positive, aerobic, spore-forming bacterium that can be misused as a biowarfare agent. The major patho-genicity factors of BA are encoded by genes located on two extrachromosomal plasmids, which are often targeted for specific identification of this pathogen. However, more recent findings show that these plasmids are not a unique feature of BA but can also occur in other Bacillus species. Furthermore, BA is a member of the Bacillus cereus group, a subgroup of closely related Bacilli. Due to the high genetic similarity within this group, it is a challenge to distinguish BA from other members of this group. In this study, we investigated if it is possible to identify species-specific and universally applicable marker peptides for BA. For this purpose, we applied a high-resolution mass spectrometry-based approach for 42 BA isolates. Together with the genomic sequencing data and by developing a bioinformatics data evaluation pipeline, which uses a database containing most of the publicly available protein sequences worldwide (UniParc), we were able to identify eleven universal marker peptides unique to BA, which are located on the chromosome and there-fore might overcome known problems like observable loss of plasmids in environmental species, plasmid loss during cultivation in the lab and that the virulence plasmids are not necessarily a unique feature of BA. The identified chromosomally encoded markers in this study could extend the small panel of already existing chromosomal targets and together with targets for the viru-lence plasmids may pave the way to an even more reliable identification of BA using genomics- as well as proteomics-based techniques.