Chlorella Beijerinck (Chlorellaceae, Trebouxiophyceae) strains from the collection of the National Institute for Environmental Studies (NIES) were characterized using gene sequence data. The misidentification of a number of strains was rectified. Chlorella vulgaris Beijerinck NIES-2173 was reclassified as C. sorokiniana Shihira et Krauss. Chlorella sp. NIES-2171 was described as a new species in the genus Micractinium Fresinius, M. inermum Hoshina et Fujiwara. Chlorella sorokiniana NIES-2167 and Chlorella sp. NIES-2330 were found to be phylogenetically related to Didymogenes Schmidle. We propose these two strains be transferred to the genus Didymogenes and given new names: D. sphaerica Hoshina et Fujiwara and D. soliella Hoshina et Fujiwara. Taxonomic decisions were primarily based on small subunit-internal transcribed spacer ribosomal DNA phylogeny for genus assignment and ITS2 sequence-structure to determine species autonomy. Our findings suggest that this strategy is the most effective way to use the species concept among autosporic coccoids.