Francisella tularensis is the etiological agent of tularemia, a zoonotic disease with worldwide prevalence. F. tularensis is a highly pathogenic organism and has been designated as a potential biothreat agent. Currently there are four recognized subspecies of F. tularensis: tularensis (type A), holarctica (type B), mediasiatica, and novicida. In addition, genomic studies have further subdivided type A tularensis into two subclassifications, type A.I and type A.II. These two subclassifications differ in geographic distribution with type A.I appearing mainly in the Eastern United States and type A.II appearing mainly in the Western United States. Because of differences of virulence among the subspecies, it is important to be able to quickly identify each of the subspecies rapidly and accurately. This work describes the development of a multiplex real-time polymerase chain reaction (PCR) assay which was shown to be ~98% successful at identifying the known subspecies of F. tularensis. Furthermore, F. tularensis is thought be a genome in decay (losing genes) because of the relatively large number of pseudogenes present in its genome. We hypothesized that the observed frequency of gene loss/pseudogenes may be an artifact of evolution in response to a changing environment, and that genes involved in virulence should be under strong positive selection. Eleven arbitrarily chosen virulence genes were screened for positive selection along with 10 arbitrarily chosen housekeeping genes. Analyses of selection yielded one housekeeping gene and 7 virulence genes which showed significant evidence of positive selection. Our results suggest that while the loss of functional genes through disuse could be accelerated by negative selection, the genome decay in Francisella could also be the byproduct of adaptive evolution, as evidenced by several of its virulence genes which are undergoing strong, positive selection.