Systematic studies on 70 MACC isolates previously identified as ‘Chlamydomonas’, a unicellular flagellate, were carried out based on partial 18S rRNA. The aim of this study was to determine the phylogenetic affiliations of Chlamydomonas strains in the MACC collection. The study found that most of the strains were not Chlamydomonas. Nine clusters of phylogenetically similar taxa were identified. The previous determinations were completed with their new phylogenetic affiliations (partly due to changes in green algae classification). Molecular data revealed that 3 of the 70 strains are from Arenicolinia, 14 are members of the phylogroup Stephanosphaerinia, 11 are Oogamochlamydinia, 1 is Chloromonadinia, 19 are Reinhardtinia, 2 are Polytominia, 9 are Scenedesmaceae, 5 are Moewusinia, and 6 are Chlorella. Clades were established by 18S rRNA similarity and p-distances. This study reveals the need to revise established culture collections whose isolates are solely identified with morphology.