In 2001, Nasutitermes corniger (Motschulsky), common name conehead termite, were discovered near a marina in Dania Beach, FL, where the invasive species was probably transported from its native range in Central and South America or the Caribbean. In January 2016, an infestation was found in Pompano Beach, Florida, approximately 21 km north of the Dania Beach population. This study compares variants in seven microsatellite loci across specimens from 11 nests in Dania Beach and 8 nests in Pompano Beach. Results are consistent with all N. corniger in both locations being descendants of a single introduced colony, spreading within Broward County, FL through human transport of infested materials. No more than four alleles were found at any of the seven microsatellite loci analyzed, inferring that a single Queen and King, or multiple sibling reproductives descended from a monogamous pair, headed the colony that arrived in Florida. The potential economic and environmental impacts of this invasive termite are enormous due to its broad diet, including agricultural crops and orchards, native and ornamental plants, natural landscapes, and structures. Conspicuous tunnels and aboveground nests are the key aspects of N. corniger biology that render colonies vulnerable to discovery and control. The now proven ability of N. corniger to establish breeding populations in the United States, to cause extensive property and landscape destruction, and to spread by human transport underscores the need for continued aggressive efforts toward eradication of known infestations as well as quick operational actions the next time invasive N. corniger are discovered.