For evolutionary comparisons, phylogenetics and evaluation of potential interbreeding taxa of a species, various loci have served for animals and plants and protistans. One [second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA] is highly suitable for all. Its sequence is species specific. It has already been used extensively and very successfully for plants and some protistans, and a few animals (where historically, the mitochondrial genes have dominated species studies). Despite initial impressions that ITS2 is too variable, it has proven to provide useful biological information at higher taxonomic levels, even across all eukaryotes, thanks to the conserved aspects of its transcript secondary structure. The review of all eukaryote groups reveals that ITS2 is expandable, but always retains in its RNA transcript a common core structure of two helices with hallmark characteristics important for ribosomal RNA processing. This aspect of its RNA transcript secondary structure can rescue difficult alignment problems, making the ITS2 a more powerful tool for phylogenetics. Equally important, the recognition of eukaryote-wide homology regions provides extensive and detailed information to test experimental studies of ribosomal rRNA processing.