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Bacillus anthracis , the aetiological agent of anthrax, is regarded as a highly monomorphic pathogen that presents a low genetic diversity using standard molecular techniques. Whole genome sequencing and single nucleotide polymorphisms (SNPs) are definitive signatures for subtyping of B. anthracis. Here we employed whole genome single nucleotide polymorphism (wgSNP) analysis to investigate the genetic diversity of B. anthracis in the historically endemic region of Northern Cape Province (NCP), South Africa. Twenty-six isolates from anthrax outbreaks that occurred between 1998 and 2008/9 in NCP as well as from Namibia-South Africa Transfontier Conservation area and Botswana were compared to global B. anthracis genomes. Most NCP B. anthracis strains (n = 22) clustered in the A.Br.003/004 (A.Br.101) branch and are closely related to the Zimbabwe and Mozambique strains (A.Br.102 branch). A total of 4923 parsimony informative-SNPs accurately established the A.Br.003/004 phylogenetic relationships of the NCP isolates into two distinct sub-clades and SNP markers designated as A.Br.172 and A.Br.173 were developed. Other NCP strains (n = 2) grouped in the A.Br.001/002 (Sterne) branch while strains (n = 2) from the Namibia-South Africa Transfontier Conservation area and Botswana clustered in A.Br.005/006 (Ancient A) branch. The sequenced B. anthracis strains (A0094, A0096 and A0097) that clustered in the A.Br.064 (V770) clade were isolated from Vaalbos National Park and similar strains have not been isolated. The B. anthracis A0088 strain cluster with the NCP strains in the A.Br.003/004 (A.Br.172) SNP branch which has been isolated in NCP, South Africa. This study highlights the phylogenetic structure of NCP B. anthracis strains with distinctive SNP branches important for forensic tracing and novel SNP discovery purposes. The sequenced strains will serve as a means to further trace the dissemination of B. anthracis outbreaks in NCP, South Africa, and on the continent, as well as for forensic tracking on a global scale. • wgSNP analysis of the Northern Cape Province, South African B. anthracis strains cluster in to new A-sub-clades. • Strains were defined by A.Br.002 (Sterne), A.Br.101, A.Br.005/006, and A.Br.V770 clades featuring unique regional SNPs. • The prevailing A.Br.101 branch is dissimilar from the A.Br.013 (Australia94 and Georgia strains) sub-clade. • Mozambique and Zimbabwe strains (A.Br.102 branch) are derived from the established A.Br.172 branch that is shared with NCP strains. [ABSTRACT FROM AUTHOR]