The limits of species ranges can be determined by a number of biotic and abiotic interactions, and in areas where closely related species overlap, some degree of reproductive isolation must exist in order for them to remain distinct. Understanding these interactions is essential for understanding what limits species distributions or causes hybridization. Fundulus heteroclitus (Mummichog) and Fundulus grandis (Gulf Killifish) are two closely related species with similar morphologies and ecological niches. Both species have widespread distributions that overlap in northeastern Florida. In the present study, two highly divergent loci (one nuclear and one mitochondrial) were utilized to distinguish these fundulid species in order to identify their ranges and to detect hybrids. Analysis of specimens collected along a north to south gradient in northeastern Florida salt marshes established that a relatively sharp transition (≈38 km) from relatively pure Mummichog populations to relatively pure F. grandis populations existed south of Jacksonville, FL, centered near Flagler Beach, FL. Putative hybrid genotypes were detected at moderate frequencies within the contact zone, suggesting that successful hybridization is likely occurring between the two species, but is relatively uncommon. These results provide a stepping stone to investigate the types of reproductive barriers that are involved in maintaining species distinctions in this system and their effects on the species' ranges and ecological interactions.