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Two green microalgal strains, CCCryo 469-16 and CCCryo 470-16, were isolated from samples of Antarctic microflora. Their morphology and 18S rRNA sequences indicated a phylogenetic relationship with strains of the genera <i>Chlorococcum</i> Meneghini, <i>Macrochloris</i> Korshikov, and <i>Pleurastrum</i> Chodat. The last genus is considered particularly problematic as it underwent several revisions. Moreover, its type strain, <i>P. insigne</i> SAG 30.93, was recently demonstrated to coincide genetically with the authentic strain of <i>Chlorococcum oleofaciens</i> from an 18S rRNA phylogeny. This deprived the genus <i>Pleurastrum</i> of an important type reference. Thus, the molecular markers <i>rbc</i>L, <i>tuf</i>A, and ITS were employed to identify the Antarctic isolates more precisely. Several other microalgae related to our isolates were obtained from international culture collections for comparison. The obtained results allowed the re-establishment of strain <i>P. insigne</i> SAG 30.93 as the reference strain and the definition of the molecular borders of both genera <i>Pleurastrum</i> and <i>Chlorococcum</i>. Based on our findings, several <i>Chlorococcum</i> species are now re-attributed to <i>Pleurastrum</i>, as well as <i>Macrochloris rubrioleum</i>, here re-named <i>Pleurastrum rubrioleum</i> comb. nov., to which the Antarctic isolates also belong. Finally, a relationship between <i>Pleurastrum</i> lineages and climate zones was established.