A molecular genetics study involving DNA sequencing of a portion of the mitochondrial DNA 16S gene was undertaken to determine the extent of genetic variation within Reticulitermes flavipes (Kollar) in North America. This study was done because differences in morphological variants (of R. flavipes) would presumably translate into genetic differences, and there are no prior studies that describe its genetic variation from the extent of its North American range. In total, 493 samples were analyzed from the United States, Canada, and Mexico. Nineteen nucleotide sites were variable in the 428-bp mitochondrial DNA (mtDNA) sequence, and 47 mtDNA haplotypes were observed. Nine haplotypes (19%) occurred only once, whereas the most common haplotype, F, occurred in 17% of the samples. Four haplotypes were found over a broad geographical range encompassing at least nine states each. The single haplotype found in Toronto, Canada, also occurs in Arkansas, whereas two of the three haplotypes found in Mexico are unique to that country. Based on this research, there seems to be numerous R. flavipes haplotypes that are widespread, perhaps due to human involvement, whereas other haplotypes may be more rare and could represent locally adapted populations.