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 the subterranean termite, Reticulitermes tibalis, in the United States. A total of 106 samples were analyzed from 18 states. Thirty-two nucleotide sites were variable in the 428 bp 16S rDNA sequence, and 41 distinct haplotypes were observed. Twenty-three haplotypes (55%) occurred only once, while the most common haplotype, T2, occurred in 24% of the samples. Genetic diversity among haplotypes ranged from 0.2 to 2.3%. Bayesian phylogenetic and TCS spanning tree analysis revealed several distinct clades that appeared to be geographically isolated; however, regression and Mantel tests did not support any populational structure. The role of glaciation and sky islands on haplotype variation of R. tibialis is supported by a molecular clock and may have contributed to the large amount of genetic variation observed within this species.