Invasive species are widely recognized as important drivers of the ongoing biodiversity crisis. The US state of Florida is especially susceptible to the proliferation of invasive reptiles, and nonnative lizards currently outnumber native lizard species. At present, there are 3 documented breeding populations of the Nile monitor (Varanus niloticus) in different regions of Southern Florida, and these populations are considered potential dangers to threatened, fossorial endemics, such as burrowing owls, American crocodiles, and gopher tortoises. Nevertheless, at present, both the introduction histories of these populations and the degree to which they are connected by gene flow are not known. To address these issues, we genotyped V. niloticus from Cape Coral, Homestead Air Reserve Base, and West Palm Beach at 17 microsatellite loci and conducted a variety of analyses to assess both intrapopulation genetic diversity, the degree of gene flow between populations, and the most likely introduction scenario. The results of our analyses demonstrate that all 3 populations have limited genetic diversity (mean number of effective alleles across loci in all 3 populations ~ 2.00) and are highly differentiated from one another (GST = 0.268; G″ST = 0.628). Our results also suggest that these populations resulted from independent introduction events that occurred within the past few decades. Consequently, we advise that wildlife managers focus management efforts on containment of existing populations and intensification of monitoring efforts on potential migration corridors.