The foliose epiphytic lichen Lobaria pulmonaria has suffered a significant decline in European lowlands during the last decades and therefore is considered as endangered throughout Europe. An assessment of the genetic variability is necessary to formulate biologically sound conservation recommendations for this species. We investigated the genetic diversity of the fungal symbiont of L. pulmonaria using 143 specimens sampled from six populations (two small, one medium, three large) in the lowland, the Jura Mountains, the pre-Alps and the Alps of Switzerland. Among all nuclear and mitochondrial regions sequenced for this study, variability was found only in the internal transcribed spacer (ITS I), with three polymorphic sites, and in the nuclear ribosomal large subunit (nrLSU), with four polymorphic sites. The variable sites in the nrLSU are all located within a putative spliceosomal intron. We sequenced these two regions for 81 specimens and detected six genotypes. Two genotypes were common, two were found only in the more diverse populations and two were found only in one population each. There was no correlation between population size and genetic diversity. The highest genetic diversity was found in populations where the fungal symbiont is reproducing sexually. Populations with low genetic diversity included only the two same common genotypes. Our study provides evidence suggesting that L. pulmonaria is self-incompatible and heterothallic. Based on our results we give populations with sexually reproducing individuals a higher rank in terms of conservation priority than strictly asexual populations. The remaining lowland populations are so small, that one single catastrophic event such as a windthrow might destroy the entire population. Hence we suggest augmenting such populations in size and genetic diversity using small thallus fragments or vegetative diaspores collected in other populations. As we did not detect any locally adapted genotypes, these transplants can be taken from any other genetically diverse population in Switzerland.