Red lionfish (Pterois volitans) have become a successful invasive predator across the Northwestern Atlantic, Caribbean, and Gulf of Mexico (GoM). Previous investigations have identified the southeast coast of Florida as the original site of introduction, but no region-wide genetic study has directly addressed the question of introduction location(s). This dataset includes previously unpublished red lionfish samples (n = 237) from six locations: The Bahamas, Florida Keys, Northwest Florida, North Carolina, Panama, and Southeast Florida. Sequences archived in NCBI from other locations in the Northern Region, Caribbean, and Gulf of Mexico basins were used in the analyses (N = 1558). Previously published sequences were obtained from Freshwater et al. (2009) (N = 396), Sealy et al. (2013) (N = 178), Whitaker and Janosik (2020) (N = 80), Butterfield et al. (2015) (N = 214), Toledo-Hernandez et al. (2014) (N = 118), Betancur-R et al. (2011) (N = 337), and Johnson et al. (2016) (N = 235). Previously unpublished North Carolina lionfish sequences were also obtained from the collection held by D. Wilson Freshwater (n = 2). Additionally, 14 microsatellite loci were multiplexed to assess connectivity and invasion pathways on 393 samples from the region.