Bactrospora dryina is an epiphytic lichen-forming fungus specifically related to old-growth floodplain forests, which have dramatically declined in Europe over the past centuries. In order to promote conservation management of such forest remnants, we aimed to study population genetics of this rare and threatened lichen. The newly developed 16 microsatellite markers are specific for the mycobiont of B. dryina and reliably amplify either single fruiting bodies or a sterile thallus. This allows the use of these markers for the identification of sterile crusts and for quantification of recent dispersal of the species into restored habitats. We tested the markers in 264 samples collected from 10 pedunculate oak trees growing in three localities in north-eastern Switzerland. All markers were polymorphic and showed two to five alleles per locus, and unbiased gene diversity varied from 0·06 to 0·71 over three populations. The relatively low number of alleles in B. dryina is possibly the consequence of colonization of secondary habitats created by forest management. Although oaks were largely covered with a single, continuous B. dryina colony, the microsatellite markers identified single or complex multi-genotype colonizations per tree. For future population genetic studies, we recommend collection of 5–15 specimens from one tree which would enable detection of 60–80% of the multilocus genotypes present. Hierarchical AMOVA revealed high variation (70%) on host trees, and a relatively high differentiation (12%) among the three locations in NE Switzerland indicated limited gene flow between those regions. Thus, the newly developed markers showed their applicability in population genetics at different spatial scales. They will play an important role in monitoring habitat restoration for the conservation of B. dryina and associated forests and riverscapes.