The Southern Ocean is one of the most isolated marine ecosystems, characterized by high levels of endemism, diversity, and biomass. Ascidians are among the dominant groups in Antarctic benthic assemblages, thus recording the evolutionary patterns of this group is crucial to improve our current understanding of the assembly of this polar ocean. We studied the genetic variation within Cnemidocarpa verrucosa sensu lato, one of the most widely distributed abundant and studied ascidian species in Antarctica. Using a mitochondrial and a nuclear gene (COI and 18S), the phylogeography of fifteen populations distributed along the Antarctic Peninsula and South America (Burdwood Bank/MPA Namuncurá) was characterized, where the bimodal distribution of the genetic distance suggested the existence of two species within the nominal C. verrucosa. When re-evaluating morphological traits to distinguish between genetically defined species, the presence of a basal disc in one of the genotypes could be a morphological trait to differentiate the species. These results are surprising due to the large research that has been carried out with the conspicuous C. verrucosa with no differentiation between species. Furthermore, it provides important tools to distinguish species in the field and laboratory. But also, these results give new insights to patterns of differentiation between closely related species that are distributed in sympatry, where the permeability of species boundaries still needs to be well understood.