The causative agent of tularemia, Francisella tularensis, is a formidable biologic agent that occurs naturally throughout North America. We examined genetic and spatial diversity patterns among 161 US F. tularensis isolates by using a 24-marker multiple-locus variable-number tandem repeat analysis (MLVA) system. MLVA identified 126 unique genotypes. Phylogenetic analyses showed patterns similar to recently reported global-scale analyses. We observed clustering by subspecies, low genetic diversity within F. tularensis subsp. holarctica, and division of F. tularensis subsp. tularensis into 2 distinct subpopulations: A.I. and A.II. The 2 F. tularensis subsp. tularensis subpopulations also represent geographically distinct groups; A.I. occurs primarily in the central United States, and A.II. occurs primarily in the western United States. These spatial distributions are correlated with geographic ranges of particular vectors, hosts of tularemia, and abiotic factors. These correlates provide testable hypotheses regarding ecologic factors associated with maintaining tularemia foci.