Trebouxiophytes of the genus Prasiola are well known in Antarctica, where they are among the most important primary producers. Although many aspects of their biology have been thoroughly investigated, the scarcity of molecular data has so far prevented an accurate assessment of their taxonomy and phylogenetic position. Using sequences of the chloroplast genes rbcL and psaB, we demonstrate the existence of three cryptic species that were previously confused under Prasiola crispa (Lightfoot) Kützing. Genuine P. crispa occurs in Antarctica; its presence was confirmed by comparison with the rbcL sequence of the type specimen (from the Isle of Skye, Scotland). Prasiola antarctica Kützing is resurrected as an independent species to designate algae with gross morphology identical to P. crispa but robustly placed in a separate lineage. The third species is represented by specimens identified as P. calophylla (Carmichael ex Greville) Kützing in previous studies, but clearly separated from European P. calophylla (type locality: Argyll, Scotland); this alga is described as P. glacialis sp. nov. The molecular data demonstrated the presence of P. crispa in Maritime and Continental Antarctica. P. antarctica was recorded from the Antarctic Peninsula and Shetland Islands, and P. glacialis from the Southern Ocean islands and coast. Such unexpected cryptic diversity highlights the need for a taxonomic reassessment of many published Antarctic records of P. crispa. The results also indicate that marine species of Prasiola form a well-supported monophyletic group, whereas the phylogenetic diversity of freshwater species is higher than previously suspected (at least three separate lineages within the genus include species living in this type of environments).