Diversity, abundance and composition of taxonomic groups in the Southern Ocean differ from elsewhere in the planet, since the biogeography in this region reflects the complex interactions of tectonics, oceanography, climate and biological elements since the Eocene. Several groups of marine benthic organisms exhibit high levels of genetic divergence among provinces in this region, supporting the existence of a vicariance process through plate tectonics, while other groups with high dispersive capacity exhibit recent divergence processes. Moreover, the discovery of non-Antarctic decapod larvae in Antarctic Peninsula suggests that some groups can travel across the Antarctic Circumpolar Current. Here we analyzed levels of genetic divergence in congeneric species of three Southern Ocean's benthic invertebrate groups with dispersive potential. For this purpose we included in the analyses COI sequences of an echinoid (Sterechinus), a gastropod (Nacella), and a bivalve (Yoldia). Considering the levels of genetic differentiation and assuming the molecular clock hypothesis we estimated the separation of invertebrates from the two continents. We also compared levels of genetic variation between Antarctic and sub-Antarctic species of Nacella and Sterechinus to determine the effect of the Quaternary glacial episodes in the demography of these species. We detected clear genetic differences between Antarctic and sub-Antarctic congeneric species of Sterechinus, Nacella, and Yoldia. According to our results, the installation of an effective barrier between Antarctica and sub-Antarctica occurred almost at the same time (between 3.7 and 5.0 Ma) for these groups of organisms, long after the physical separation of both continents. Genetic comparisons between Antarctic and Sub-Antarctic species of Nacella and Sterechinus detected lower levels of genetic diversity in Antarctic species, suggesting more pronounced effects of the glacial episodes in Antarctica than in South America. These results may reflect the dramatic effect of the Quaternary glacial cycles on Antarctic population sizes, especially in groups with narrow bathymetric ranges. The present study provides new evidence about the differentiation processes between Antarctic and South American organisms. None of the analyzed genera showed evidence for recurrent gene flow across the Antarctic Circumpolar Current since the Mio-Pliocene. Genetic comparisons indicate that Antarctic and Sub-Antarctic species were differentially affected by glacial periods.