Sequencing of the mitochondrial DNA cytochrome oxidase II (COII) gene was undertaken to determine genetic variation and distribution of the subterranean termite Reticulitermes in two sites separated by more than 200 km in Georgia (USA). Four hundred samples were collected from the field every month from 2002 to 2005 and preserved in alcohol. A 584 base pair region of the mtDNA COII gene was amplified from an individual termite from each sample in both directions using polymerase chain reaction (PCR) and sequenced. The sequence data was used to defined 19 maternal lineages. Eighty polymorphic sites were identified among these lineages. Termites which shared one maternal lineage were collected from disparately distant sites. Phylogenies for these sequences were estimated by neighbor-joining and maximum-parsimony analysis. Both methods resulted in trees with highly similar topologies. Node support resulted from 1000 bootstrap pseudoreplicates or 100 nonparametric pseudoreplicates. Most of the maternal lineages were very close which indicate that the termites of these lineages could share a common maternal ancestor. However, other lineages were very different which likely represent different species. Information on Reticulitermes gene flow will help to facilitate development of novel treatment strategies and a better understanding of the social and ecological factors affecting gene flow.