The 16S ribosomal DNAs (rDNAs) of two strains of Francisella tularensis and one strain of Francisella philomiragia were sequenced. On the basis of phylogenetic analysis data, the genus Francisella was placed in the gamma subclass of the Proteobacteria. The most closely related organism was the intracellular bacterium Wolbachia persica. The sequenced 16S rDNA molecules of the Francisella species exhibited very high levels of similarity (98.5 to 99.9%). Two variable regions, comprising 390 to 450 nucleotides of the 16S rDNA molecules of 17 additional Francisella strains, including members of the species F. tularensis and F. philomiragia, were also sequenced. At most, six nucleotide differences were observed among the sequences of the F. tularensis strains. The sequence of Francisella novicida was virtually identical to the sequences of the F. tularensis strains, thereby supporting the hypothesis that these organisms are members of the same species. On the basis of the observed differences, primer pairs were designed to distinguish strains by using the PCR at the genus, species, and subspecies levels. This permitted sensitive identification of strains belonging to the genus Francisella and discrimination of the species F. tularensis and F. philomiragia.