Intrathalline phycobiont diversity was investigated in a rosette-forming lichen, Parmotrema pseudotinctorum, using a combination of Sanger sequencing, 454-pyrosequencing, conventional light and confocal microscopy, and transmission electron microscopy. A total of 39 thalli sampled in five Canary Island populations were investigated. Three novel lineages of lichen phycobionts were detected, all being inferred within the Trebouxia clade G. The most abundant phycobiont lineage, occurring in all lichen populations investigated, is described here as Trebouxia crespoana sp. nov. This species produces spherical to pyriform cells possessing a crenulate chloroplast with lobes elongated at their ends, and one corticola-type pyrenoid with very thin, unbranched tubules of curved profile. Trebouxia crespoana is clearly distinguished from all other Trebouxia species by a characteristic cap-like cell wall thickening produced on one side of vegetative cells, and the larger size of vegetative cells that reach 21(–26) µm in diameter.