Francisella tularensis is a bacterial pathogen that causes the zoonotic disease tularemia and is important to biodefense. Currently, the only vaccine known to confer protection against tularemia is a live vaccine strain (named LVS) derived from a Francisella tularensis ssp. holarctica virulent isolate. The origin and source of attenuation of this strain are not known. To assist with the design of a defined live vaccine strain, we sought to determine the genetic basis of the attenuation of LVS. This analysis relied primarily on the comparison between the genome of LVS and the Francisella tularensis holarctica strain FSC200, which differ by only 0.08% of their nucleotide sequence. Under the assumption that the attenuation was due to a loss of function(s), only coding regions were examined in this comparison. To complement this analysis, the coding regions of two slightly more distantly related Francisella tularensis strains were also compared against the LVS coding regions. Thirty-five genes show unique sequenc