The distribution of polyols was examined for the first time in 34 green algal strains from four different clades belonging to the class Trebouxiophyceae, which dominate aeroterrestrial biofilms of many regions. Sorbitol was detected in representatives of the Prasiola clade, while ribitol was present in the Elliptochloris and Watanabea clades. Apatococcus lobatus (Chodat) J. B. Petersen contained erythritol in addition to ribitol. Polyols are considered as effective stress metabolites exerting multiple protective functions in metabolism and hence mainly occur in organisms colonizing extreme environments. In contrast, members of the Chlorella clade, which mainly occur in aquatic habitats, did not contain polyols. Thus, the constitutive presence of specific polyols facilitates a differentiation between species of the Prasiola clade from the Elliptochloris and Watanabea clades, respectively, and further allows differentiation of morphologically converging taxa.