The dwarf seahorse (Hippocampus zosterae) is widely distributed throughout near-shore habitats of the Gulf of Mexico and is of commercial significance in Florida, where it is harvested for the aquarium and curio trades. Despite its regional importance, the genetic structure of dwarf seahorse populations remains largely unknown. As an aid to ongoing conservation efforts, we employed three commonly applied mtDNA markers (ND4, DLoop and CO1) to investigate the genetic structuring of H. zosterae in Florida using samples collected throughout its range in the state. A total of 1450 bp provided sufficient resolution to delineate four populations of dwarf seahorses, as indicated by significant fixation indices. Despite an overall significant population structure, we observed evidence of interbreeding between individuals from geographically distant sites, supporting the hypothesis that rafting serves to maintain a degree of population connectivity. All individuals collected from Pensacola belong to a single distinct subpopulation, which is highly differentiated from the rest of Floridian dwarf seahorses sampled. Our findings highlight the utility of mtDNA markers in evaluating barriers to gene flow and identifying genetically distinct populations, which are vital to the development of comprehensive conservation strategies for exploited taxa.