The taxonomy of Vitreochlamys species (Volvocales, Chlorophyta) was studied with light and electron microscopy; DNA sequence data were obtained for nine strains from various localities in Japan, the Czech Republic, Slovak Republic and Germany. The strains could be delineated clearly into six species – V. fluviatilis (Stein) Batko, V. gloeocystiformis (Dill) Nakazawa, comb. nov., V. ordinata (Skuja) Nakazawa, comb. nov., V. nekrassovii (Korshikov) Nakazawa, comb. nov., V. aulata (Pascher) Batko, and V. pinguis Nakazawa, sp. nov. – on the basis of differences in cell and protoplast shape, numbers of contractile vacuoles and pyrenoids, form and position of the stigma, location of the nucleus, degree of longitudinal striations on the chloroplast surface, and ultrastructure of the pyrenoid and stigma. Although V. pinguis and V. aulata share common chloroplast morphology, including stigma and pyrenoid ultrastructure, these two species can be distinguished clearly by cell shape, the number of contractile vacuoles and location of the nucleus. In addition, phylogenetic sequence data of the rbcL (large subunit of Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase) gene from all of the Vitreochlamys strains strongly suggest that V. pinguis from Japan is robustly separated from the clade composed of four strains of V. aulata from the Czech Republic, Slovak Republic and Japan. The sequence data also showed that Vitreochlamys is composed of three clades, which are largely consistent with the ultrastructural features of the stigma and pyrenoids.