This study was the first to use DNA sequencing data to infer the origin of a Formosan subterranean termite, Coptotermes formosanus Shiraki, infestation. Cytochrome oxidase II gene sequence was used to define eight specific maternal lineages from 14 geographic locations across the southeastern United States, Hawaii, and China. One maternal lineage, A, was unique to all the Atlanta collections and one New Orleans collection, which indicated that termites at these sites shared a common maternal ancestor. The impact of interstate commerce in terms of Formosan subterranean termite introductions is addressed as is the industry response needed to arrest the spread of these termites via commercial traffic in used railroad cross ties.