This paper is the first detailed description of the development and use of new genetic tools specifically for the safe manipulation of highly pathogenic Francisella tularensis subsp. tularensis. Most of these tools are also demonstrated to work with other F. tularensis subspecies. Kanamycin and hygromycin resistance determinants that function as genetic markers in F. tularensis subsp. tularensis strain Schu and sets of episomal shuttle vectors that are either unstable or stably maintained in the absence of selection were developed. In addition, the hyg gene, expressed from the F. tularensis groESL promoter, was successfully used as a marker for transposon mutagenesis. This work also includes the development of sacB-based suicide plasmids expressing kanamycin resistance that can be used for electroporation-mediated allelic exchange of unmarked mutations in Schu and the F. tularensis live vaccine strain (LVS). Using these plasmids, the two predicted beta-lactamase genes, blaA and blaB, in Schu and LVS were deleted. Only the Delta blaB1 mutants had increased susceptibility to ampicillin, and this phenotype was complemented by a plasmid expressing blaB+. The results suggest that the beta-lactam antibiotic resistance phenotype of Schu and LVS is likely due to only one of the two beta-lactamase genes present and that ampicillin resistance can be used as an additional selectable marker in beta-lactamase deletion mutants. The collection of tools presented in this report will be helpful for the genetic analyses of F. tularensis subsp. tularensis pathogenesis.