The red lionfish Pterois volitans is a successful invasive predator across the western North Atlantic, Caribbean, and Gulf of Mexico. The southeast coast of Florida (USA) has been identified as the original introduction location, but genetic analyses including Florida lionfish have yet to investigate introduction scenarios. Here, we assessed the potential lionfish invasion pathways using 1795 sequences from previously published mitochondrial D-loop sequences (n = 1558) and new samples (n = 237) from 6 locations: The Bahamas, Florida Keys, northwest Florida, North Carolina, Panamá, and southeast Florida. None of the assessed Florida lionfish (n = 394) contained the H05-H09 D-loop haplotypes found in The Bahamas, North Carolina, and Bermuda (the Northern Region), indicating that Florida was not the source for these haplotypes. Assessing the mitochondrial population structure, the Florida east coast lionfish grouped with the Caribbean/Gulf of Mexico, as opposed to the Northern Region. To further explore connectivity and invasion pathways, 14 nuclear microsatellite loci were multiplexed on lionfish collected from 15 locations (n = 394). As found in other nuclear lionfish studies, the analyses identified a lack of population structure likely due to founding effects and/or inbreeding in aquaculture brood stocks. Together, the significant haplotype differences and H01-H04 haplotypes refute Florida as the sole source of red lionfish introduction. The results of this study support alternative invasion scenarios, in which Florida was colonized as a secondary introduction site or by individuals from the Northern Region. Understanding invasive species’ population boundaries and dispersal patterns informs local control efforts and management planning for future invasive species introductions.