Francisella tularensis is a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies tularensis, mediaasiatica, holarctica, and novicida. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent F. tularensis subspecies tularensis, strain Schu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species F. tularensis, and when strains were compared, at most 3.7 % of the probes showed differential hybridization. Despite marked differences in their virulence and geographical origin, a high genomic similarity between strains of the subspecies tularensis and mediaasiatica was apparent. Within the subspecies holarctica, strains from Japan showed unique hybridization patterns. Eight Regions of Difference (RD), 0.6 - 11.5 kb in size altogether comprising 21 open reading frames, were identified that distinguished strains of the moderately virulent subspecies holarctica and the highly virulent subspecies tularensis, respectively. One of these regions, RD1, allowed for the first time the development of a F. tularensis specific PCR assay discriminating each of the four subspecies.