The nature of Chlorella symbioses in invertebrates and protists has attracted much interest, but the uncertain taxonomy of the algal partner has constrained a deeper ecological understanding of this symbiosis. We sequenced parts of the nuclear 18S rDNA, the internal transcribed spacer (ITS)-1 region, and the chloroplast 16S rDNA of several Chlorella isolated from pelagic ciliate species of different lakes, Paramecium bursaria symbionts, and free-living Chlorella to elucidate phylogenetic relationships of Chlorella-like algae and to assess their host specificity. Sequence analyses resulted in well-resolved phylogenetic trees providing strong statistical support for a homogenous ‘zoochlorellae’ group of different ciliate species from one lake, but clearly different Chlorella in one of those ciliate species occurring in another lake. The two Chlorella strains isolated from the same ciliate species, but from lakes having a 10-fold difference in underwater UV transparency, also presented a distinct physiological trait, such as the ability to synthesize UV-absorbing substances known as mycosporine-like amino acids (MAAs). Algal symbionts of all P. bursaria strains of different origin resolved in one clade apart from the other ciliate symbionts but split into two distinct lineages, suggesting the existence of a biogeographic pattern. Overall, our results suggest a high degree of species specificity but also hint at the importance of physiological adaptation in symbiotic Chlorella.