A multimethod approach was used to characterize unicellular green algae that were traditionally assigned to the genus Chlorella Beijerinck and to resolve their phylogenetic relationships within the Chlorophyta. Biochemical, physiological, and ultrastructural characters, together with molecular data such as DNA base composition and DNA hybridization values, were compared with a molecular phylogeny based on complete 18S rRNA sequences. Our results show that Chlorella taxa are dispersed over two classes of chlorophytes, the Trebouxiophyceae and the Chlorophyceae. We propose that only four species should be kept in the genus Chlorella (Chlorophyta, Trebouxiophyceae): C. vulgaris Beijerinck, C. lobophora Andreyeva, C. sorokiniana Shih. et Krauss, and C. kessleri Fott et Nováková. Common characteristics of these taxa are glucosamine as a dominant cell wall component and the presence of a double thylakoid bisecting the pyrenoid matrix. Norspermine, norspermidine, and secondary carotenoids are never produced. Other “Chlorella” species belong to different taxa within the Trebouxiophyceae (“C.” protothecoides=Auxenochlorella protothecoides[Krüger] Kalina et Punčochářová, “C.” ellipsoidea, “C.” mirabilis, “C.” saccharophila, and “C.” luteoviridis) and Chlorophyceae (“C.” zofingiensis and “C.” homosphaera=Mychonastes homosphaera Kalina et Punčochářová). The latter taxa can easily be recognized by the production of secondary carotenoids under nitrogen-deficient conditions.