Lyme disease, caused by the bacterium Borrelia burgdorferi sensu stricto, is an emerging zoonotic disease in Canada and is vectored by the blacklegged tick,Ixodes scapularis. Here we used Bayesian analyses of sequence types (STs), determined by multilocus sequence typing (MLST), to investigate the phylogeography of B. burgdorferi populations in southern Canada and the United States by analyzing MLST data from 564 B. burgdorferi-positive samples collected during surveillance. A total of 107 Canadian samples from field sites were characterized as part of this study, and these data were combined with existing MLST data for samples from the United States and Canada. Only 17% of STs were common between both countries, while 49% occurred only in the United States, and 34% occurred only in Canada. However, STs in southeastern Ontario and southwestern Quebec were typically identical to those in the northeastern United States, suggesting a recent introduction into this region from the United States. In contrast, STs in other locations in Canada (the Maritimes; Long Point, Ontario; and southeastern Manitoba) were frequently unique to those locations but were putative descendants of STs previously found in the United States. The picture in Canada is consistent with relatively recent introductions from multiple refugial populations in the United States. These data thus point to a geographic pattern of populations of B. burgdorferi in North America that may be more complex than simply comprising northeastern, midwestern, and Californian groups. We speculate that this reflects the complex ecology and spatial distribution of key reservoir hosts.