The definition of species plays a pivotal role in biology. It has been proposed that Compensatory Base Changes (CBCs) in the fast-evolving Internal Transcribed Spacer 2 (ITS2) correlate with speciation and thus can be used to distinguish species. The applicability of CBC - based species concepts using ITS2, however, rests on the homology of the investigated ITS2 positions. We studied the ITS2 molecule of 147 strains of Chlorophyceae (Chlorophyta, Viridiplantae) including 26 new sequences in the order Chaetophorales, and compared their secondary structures to ITS2 in the sister class Ulvophyceae, represented by the order Ulvales. Using a phylogenetic/comparative approach, it was possible to identify 1) the first consensus structure model of the ITS2 molecule that can be applied to two classes of green algae [Ulvophyceae (Ulvales), Chlorophyceae] and 2) landmarks (the spacer regions separating the ITS2 Helices) for more robust prediction of the secondary structures in green algae. Moreover, we found that CBCs in homologous positions in these 147 strains (representing 115 validly described species) are either completely absent or mostly associated with internal branches representing higher order taxonomic levels (genera, families, orders). As reported for the Ulvales, CBCs are not diagnostic at the species level in the dataset used.