Genotyping of Ixodes scapularis Say (Acari: Ixodidae) ticks could enhance understanding of the occurrence and genotypes of I. scapularis-borne pathogens. We investigated the utility of mitochondrial (mt) Cytochrome C Oxidase subunit I gene (cox1) sequences as a tool for understanding the population structure of I. scapularis collected in Canada, where we also investigated the geographic occurrence of different cox1 haplotypes. Sequences obtained from 414 ticks were one of 55 unique haplotypes, most of which grouped into one of six clades. Demographic analysis suggested that cox1 sequences have haplotype and nucleotide diversity comparable to other mt genes. All haplotypes were connected in a single minimum spanning network tree. Despite low fixation index values there were significant differences in the frequency of occurrence of haplotypes of different clades among four geographic regions: 1) Alberta to western Ontario, 2) eastern Ontario, 3) Quebec, and 4) Atlantic Provinces; suggesting that cox1 sequences could reveal population structure differences between I. scapularis in geographically separated populations of northeastern and midwestern North America. Spatial clusters of ticks of the same haplotype identified in regions of southern Quebec and southern Ontario where I. scapularis is invading were consistent with population bottlenecks associated with founder events. These findings suggest that cox1 sequences are useful for the study of I. scapularis population structure, are of sufficient diversity that spatial analyses of haplotypes can be used to identify where I. scapularis is emerging in southern Canada, and may be useful for exploring differences between northeastern and midwestern populations of I. scapularis.