Francisella tularensis is a gram-negative organism found in many regions of the world. F. tularensis can cause a fatal, febrile illness, although these natural tularemia infections are rare in the United States. However, the development of F. tularensis as a potential weapon of bioterrorism during the Cold War spurred the development of a live attenuated vaccine, LVS, from F. tularensis subsp. holarctica in the 1960s. Two colony morphology variants, LVS-G and LVS-R, were generated from parental LVS by plate passage and by acridine orange mutagenesis, respectively. In vaccinated mice, LVS-G and LVS-R exhibit altered immunogenicity and protective capacities. While the exact nature of the mutations in these strains was unknown, previous studies indicated that both had altered lipopolysaccharide structures. To better understand the impact of these mutations on LVS' immunogenicity, we sequenced the genomes of LVS-G and LVS-R as well as our parental laboratory stock of LVS, originally obtained from ATCC, and compared these to the F. tularensis subsp. holarctica LVS genome currently deposited in GenBank. The results indicate that the genomic sequence of ATCC LVS is nearly identical to that of the human LVS vaccine. Furthermore, a limited number of genomic mutations likely account for the phenotypes of LVS-G and LVS-R.