Studying the secondary structure of ITS2 is a key tool in molecular systematics, expanding the capabilities of primary nucleotide sequence analysis. In most eukaryotes, ITS2 forms a conserved pattern of four helices, I–IV. An accurate model of this structure is critical for phylogeny and species delimitation. Analysis of compensatory substitutions (CBCs) in ITS2 is a necessary element of the species identification algorithm. Studies of various taxonomic groups of green microalgae have revealed potential problems and limitations of using CBCs in analysis. Modeling the secondary structure of ITS, searching for CBCs, and understanding the evolutionary history of specific taxa of microalga are important aspects of species identification.