The genus Trebouxia includes the most widespread green microalgae inhabiting lichen thalli worldwide. Nonetheless, the vast majority of species-level phylogenetic lineages within this genus lack a formal taxonomic description and, to date, only 31 species have been accepted. In this study, we utilized an integrative taxonomic approach to describe the new species Trebouxia barrenoae Pazos, Chiva, Garrido-Benavent et Moya, a frequent symbiont in different lichens which is phylogenetically circumscribed to Trebouxia clade S. The ultrastructural features of the new microalga were studied through light, confocal, and electron microscopies; phylogenetic, physiological, and ecological niche analyses were carried out to characterize this new taxon as well. Our findings revealed a more frequent association of T. barrenoae sp. nov. with vegetatively reproducing lichens that thrive in temperate forest habitats and showed a preference for those growing on acidic substrates. The description of this species fosters progress in the systematics and taxonomy of the genus Trebouxia and enhances precise characterization of lichen phycobionts. Keywords: chlorophyll fluorescence, epiphytic lichen, niche hypervolume, photobiont, symbiosis, taxonomic integrative approach, transmission electron microscopy (TEM).