The field of microbiology was established in the 17(th) century upon the discovery of microorganisms by Antonie van Leeuwenhoek using a single-lens microscope. Now, the detailed ultrastructures of microorganisms can be elucidated in situ using three-dimensional electron microscopy. Since the availability of electron microscopy, the taxonomy of microscopic organisms has entered a new era. Here, we established a new taxonomic system of the primitive algal genus Glaucocystis (Glaucophyta) using a new-generation electron microscopic methodology: ultra-high-voltage electron microscopy (UHVEM) and field-emission scanning electron microscopy (FE-SEM). Various globally distributed Glaucocystis strains were delineated into six species, based on differences in in situ ultrastructural features of the protoplast periphery under UHVEM tomography and in the mother cell wall by FE-SEM, as well as differences in the light microscopic characteristics and molecular phylogenetic results. The present work on Glaucocystis provides a model case of new-generation taxonomy.