Recent data revealed that metazoans such as mites and springtails have persisted in Antarctica throughout several glacial-interglacial cycles, which contradicts the existing paradigm that terrestrial life was wiped out by successive glacial events and that the current inhabitants are recent colonizers. We used molecular phylogenetic techniques to study Antarctic microchlorophyte strains isolated from lacustrine habitats from maritime and continental Antarctica. The 14 distinct chlorophycean and trebouxiophycean lineages observed point to a wide phylogenetic diversity of apparently endemic Antarctic lineages at different taxonomic levels. This supports the hypothesis that long-term survival took place in glacial refugia, resulting in a specific Antarctic flora. The majority of the lineages have estimated ages between 17 and 84 Ma and probably diverged from their closest relatives around the time of the opening of Drake Passage (30-45 Ma), while some lineages with longer branch lengths have estimated ages that precede the break-up of Gondwana. The variation in branch length and estimated age points to several independent but rare colonization events.