The blacklegged tick, Ixodes scapularis Say, is the principal vector of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner east of the Rocky Mountains. The population genetic structure was determined using DNA sequences of a 253-bp region of the mitochondrial DNA rRNA 16S gene from 46 blacklegged ticks collected from canines, Canis lupus familiaris L., and white-tailed deer, Odocoileus virginianus (Zimmermann), in four Arkansas ecoregions. Of the 253 nucleotide characters, 21 were variable. Eighteen haplotypes were identified of which 11 occurred only once. Most of the sequences were haplotype I×9 (30%) which was found in all three ecoregions and on both host species. Molecular phylogenetic relationship of the 18 blacklegged tick haplotypes was constructed using other Ixodes species as outgroup taxa, and two I. scapularis clades were found representing the American and Southern lineages. Our results indicated that blacklegged tick is genetically variable in Arkansas, and the presence of the American lineage increases the risk for Lyme disease in Arkansas.