Lichen-forming fungi are symbiotic organisms forming a thallus with autotrophic green algae and/or cyanobacteria. Protoparmeliopsis muralis (Schreb.) Choisy is a green-algal lichen-forming fungus associating with Trebouxia photobionts. It is known as one of the most successful urban lichens in the world. In this paper, the development of microsatellite markers specific for the mycobiont of Protoparmeliopsis muralis is reported. In order to avoid algal contaminations, the pure mycobiont culture was obtained and subsequently used for DNA isolation. For DNA enrichment, the Fast Isolation by AFLP of Sequences Containing Repeats (FIASCO) method was applied. Of the 380 clones sequenced, 62 contained repeats. In total, 38 primer pairs were designed and tested, and finally 7 primer pairs were polymorphic based on 21 specimens of P. muralis.