It has only recently been possible to detect sufficient genetic diversity among anthrax isolates to allow genotype grouping (Keim et al. 1997). Early results of such grouping suggest that the southern African subcontinent may be the geographical origin of Bacillus anthracis. This report describes a pilot investigation of the genetic diversity of a study group of isolates from the Kruger National Park, South Africa, and efforts to detect spatio-temporal clustering within the study group. This study has also served as further validation for the newly developed Multi-Locus VNTR Analysis (MLVA), designed to simplify genotyping of B. anthracis isolates. The results reveal a diverse range of genotypes within the park allied with three genotype reference groups, and show that the MLVA procedure is robust for rapid analysis of B. anthracis genotypes. We also observed multiple genotype groups within epidemics and between geographically and temporally close epidemic episodes. This is in contrast to earlier characterizations of anthrax epidemics. The result of a Mantel test for time-space clustering indicates clustering of the anthrax isolates selected for the study.