We tested the utility of a 230 base pair intron fragment of the highly conserved nuclear gene Elongation Factor 1-alpha (EF1-α) as a proper marker to reconstruct the phylogeography of the marine amphipod Pontogammarus maeoticus (Sowinsky, 1894) from the Caspian and Black Seas. As a prerequisite for further analysis, we confirmed by Southern blot analysis that EF1-α is encoded at a single locus in P. maeoticus. We included 15 populations and 60 individuals in the study. Both the phylogeny of the 27 unique alleles found and population genetic analyses revealed a significant differentiation between populations from the aforementioned sea basins. Our results are in remarkable agreement with recent studies on a variety of species from the same area, which invariably support a major phylogeographic break between the Caspian and Black Seas. We thus conclude that our EF1-α intron is an informative marker for phylogeographic studies in amphipods at the shallow population level.