Specimens of Xanthoria parietina were collected from worldwide locations and ascospore discharge used to establish axenic cultures of the mycobiont. DNA was extracted and RAPD-PCR fingerprinting of 59 isolates was successfully achieved, resulting in 58 unique fingerprints. 110 multilocus RAPD markers were generated and used to construct a dendrogram. Two main groups were distinguished (75% bootstrap support): the first comprising samples from the Iberian Peninsula, the Balearic and Canary Islands; the second comprising all other worldwide samples including isolates from throughout Europe and North America. Samples from Australia and New Zealand clustered with the second group except one additional, phenotypically distinct specimen, not belonging to X. parietina, which formed an outgroup. However, comparative DNA sequence analyses are required to verify this interpretation.