The term phycobiome was recently introduced to designate all the microalgae (primary or non-primary) associated with lichen symbioses. Abundant non-primary symbiotic microalgae are usually obtained from lichen isolations, confirming that thalli are a source of biodiversity and new species. In this study, microalgae were isolated from thalli of Buellia zoharyi, Ramalina farinacea and Parmotrema pseudotinctorum collected in the Iberian Peninsula and the Canary Islands. Excluding Trebouxia phycobionts, 17 strains similar to Stichococcus (Prasiola clade) were obtained. Molecular identification was carried out by nuclear ITS sequencing, and a phylogenetic tree was generated from these sequences, and grouping them into 4 clades: Diplosphaera chodatti, Diplosphaera sp.1. Deuterostichocuccus sp.1. and Tritostichococcus coniocybes. It is also noteworthy that Diplosphaera sp.1 was detected and isolated from three phylogenetically distant lichenized fungi (B. zoharyi, R. farinacea and P. pseudotinctorum), which were sampled in ecologically different localities, namely Tenerife, La Gomera and Castellón. These results reinforce the idea of the constant presence of certain microalgae associated with the lichen thalli which, despite not being the main primary photobiont, probably form part of the lichen’s phycobiomes. Keywords : Isolation · Phylogeny · Prasiola clade Trebouxiophyceae.