We isolated 12 microsatellite loci for the epiphytic lichen-forming ascomycete Lobaria pulmonaria and studied their patterns of variation within and among populations from Canada and Switzerland. Even though several microsatellites exhibited high levels of variability at different spatial scales, we did not find any evidence for intrathalline variation. Most of the genetic variation was attributed to differences among individuals within populations. High genetic variation was also detected among L. pulmonaria samples taken from individual trees, suggesting that either multiple colonization events had occurred or that local recombination is frequent. The geographically structured distribution of alleles from several microsatellites indicated that L. pulmonaria from Canada and Switzerland represent two distinct evolutionary lineages. The potential to identify multiple alleles, and their transferability to closely related species, make microsatellites an ideal tool to study dispersal, population differentiation, and microevolution in lichens.