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Francisella tularensis is ubiquitous in the Northern Hemisphere. Yet, little is known about the disease and its ecology within Canada as few serological studies have shown exposure to the disease and fewer case studies have been reported. This report is the first to describe the molecular subtyping of F. tularensis isolates within eastern Canada using multiple-locus variable-number tandem-repeat analysis. From 1998 to 2011, a total of 73 specimens were isolated from unique human and animal sources. As expected, F. tularensis subsp. tularensis AI and F. tularensis subsp. holarctica subtypes were observed, corresponding to the known geographical division within this species. The majority of human isolates (78%) and all animal (hare) isolates were of the more virulent, AI type. Half of the B isolates were isolated from patients living in a region of Quebec where muskrat densities are known to be high. A relatively high level of marker diversity was found, suggestive of multiple introductions of the organism to the region, or more likely ongoing endemicity. There was no evidence of ongoing outbreaks or transmission, and the bulk of cases were likely due to interaction between human activity and the environment (e.g. hunting/trapping activities).This study reveals the diversity of Francisella tularensis in eastern Canada using multiple-locus variable-number tandem-repeat analysis. It was initiated to further the understanding of the species within North America as previous studies elucidating the diversity and phylogeography of the species have consisted mostly of specimens from the United States. Type A tularaemia, the most life-threatening subtype of the species and a Category A biothreat agent, is restricted to North America, and this study serves to broaden the knowledge of the epidemiology and diversity of the organism.