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<i>Buellia zoharyi</i> is a crustose placodioid lichen, usually occurring on biocrusts of semiarid ecosystems in circum-Mediterranean/Macaronesian areas. In previous work, we found that this lichenized fungus was flexible in its phycobiont choice in the Canary Islands. Here we test whether geography and habitat influence phycobiont diversity in populations of this lichen from the Iberian Peninsula and Balearic Islands using Sanger and high throughput sequencing (HTS). Additionally, three thallus section categories (central, middle and periphery) were analyzed to explore diversity of microalgal communities in each part. We found that <i>B. zoharyi</i> populations hosted at least three different <i>Trebouxia</i> spp., and this lichen can associate with distinct phycobiont strains in different habitats and geographic regions. This study also revealed that the <i>Trebouxia</i> composition of this lichen showed significant differences when comparing the Iberian Peninsula with the Balearics thalli. No support for differences in microalgal communities was found among thallus sections; however, several thalli showed different predominant <i>Trebouxia</i> spp. at each section. This result corroborate that thallus parts selected for DNA extraction in metabarcoding analyses are key to not bias the total phycobiont diversity detected. This study highlights that inclusion of HTS analysis is crucial to understand lichen symbiotic microalgal diversity.