DNA sequence variation at a mitochondrial and a nuclear intron locus was surveyed within and among multiple populations of the inland silverside (Menidia beryllina) from the southeastern United States and revealed discordant phylogenetic patterns but similar patterns of population genetic variation across nuclear and mitochondrial loci. Mitochondrial variation was geographically structured, with strongly supported monophyletic assemblages among Gulf of Mexico population samples and a close association of the St John's River (SJ) population with these same samples. Nuclear alleles were not strongly structured geographically, with little support for monophyly within or across basins. Conversely, population genetic parameters indicate that the bulk of genetic diversity for both genomes resides within and among Gulf of Mexico populations and that diversity within the Atlantic is largely restricted to the SJ population. The contrast in genetic variation and population phylogenies appears to be a function of historical demographic processes, most likely directed by fluctuating geomorphology of the Florida peninsula in response to North American glaciation cycles.