Sequencing of the mitochondrial DNA cytochrome oxidase II (mtDNA COII) gene was undertaken to determine genetic variation, distribution and colony organization of the subterranean termite Reticulitermes in four sites of 190 m2 each in Sapelo Island (Georgia, USA). Three thousands samples were collected from the field every month from 2004 to 2006 and preserved in alcohol. A 716 base pair region of the mtDNA COII gene was amplified from one to five termites from each sample in both directions using polymerase chain reaction and sequenced. The sequence data was used to define 23 maternal lineages. One hundred and seven polymorphic sites were identified among these lineages. Phylogenies for these sequences were estimated by neighbor-joining, maximum likelihood and maximum-parsimony analysis. All methods resulted in trees with highly similar topologies. Eighteen of the maternal lineages had between 2-3 base pairs that were different from a single consensus sequence. However, other lineages provided up to 50 differences and likely represent different species. According to phylogenies and mandibular morphology of soldiers, two species have been identified in this study (R. flavipes and R. virginicus). Also, an unidentified species has been reported. This investigation revealed new information about movement of termite populations in a wildland habitat and interactions between termite populations.