New World screwworm (NWS), Cochliomyia hominivorax (Coquerel 1858) (Diptera: Calliphoridae), is a myiasis-causing fly that can be a serious threat to the health of livestock, wildlife, and humans. Its progressive eradication from the southern United States, Mexico, and Central America from the 1950s to 2000s is an excellent example of successful pest management using sterile insect technique (SIT). In late 2016, autochthonous NWS were detected in the Florida Keys, representing this species' first invasion in the United States in >30 yr. Rapid use of quarantine and SIT was successful in eliminating the infestation by early 2017; however, the geographic source of this infestation remains unknown. Here, we use amplicon sequencing to generate mitochondrial and nuclear sequence data representing all confirmed cases of NWS from this infestation, and compare these sequences to preexisting data sets sampling the native distribution of NWS. We ask two questions regarding the FL Keys outbreak. First, is this infestation the result of a single invasion from one source, or multiple invasions from different sources? And second, what is the geographic origin of this invasion? We found virtually no sequence variation between specimens collected from the FL Keys outbreak, which is consistent with a single source of introduction. However, we also found very little geographic resolution in any of the data sets, which precludes identification of the source of this outbreak. Our lack of success in answering our second question speaks to the need for finer-scale genetic or genomic assessments of NWS population structure, which would facilitate source determination of potential future outbreaks.