The Tampa Bay region of Florida (USA) is a hot spot for non-native freshwater fishes. However, published information on most non-native fishes in the basin is not current. Systematic sampling efforts targeting non-native fishes in the region were conducted from 2013–2015 by the University of Florida Tropical Aquaculture Laboratory. Data from these recent surveys were analyzed, along with historic and new data from published and unpublished sources, to assess current fish distributions and determine status. We focus on five of the non-native species sampled: pike killifish Belonesox belizanus Kner, 1860, green swordtail Xiphophorus hellerii Heckel, 1848, southern platyfish Xiphophorus maculatus (Günther, 1866), Mayan cichlid Mayaheros urophthalmus (Günther, 1862), and Jack Dempsey Rocio octofasciata (Regan, 1903). All five were found to have reproducing populations in the basin, each showing broader distributions than previously indicated. Non-native populations of four of the species have persisted in the Tampa Bay region since at least the 1990s. In contrast, the presence of Mayan cichlid in the basin was not confirmed until 2004. Based on numbers, distributions, and years of persistence, these five species all maintain established populations. Pike killifish and Mayan cichlid are established and spreading throughout multiple habitat types, while green swordtail, southern platyfish, and Jack Dempsey are localized and found primarily in more marginal habitats (e.g., small ditches and first order tributary streams). Factors affecting continued existence and distributions likely include aquaculture, biotic resistance, and thermal and salinity tolerances. We also clarify non-native species status determination using a multi-agency collaborative approach, and reconcile differences in terminology usage and interpretation.