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)