This paper describes our investigation of genetic variation and clonal structure of the Mediterranean moss Pleurochaete squarrosa (Brid.) Lindb. (Pottiaceae), using inter-simple sequence repeat (ISSR) molecular markers and trnL(UAA) (intron of plastid gene for Leu tRNA) sequence, choosing different sampling strategies and scales on 16 European populations. Moreover, the intercontinental distribution of two trnL haplotypes, previously found over a large area and including 24 populations in three continents, was also investigated. Despite the prevalent asexual reproduction, P. squarrosa shows a high level of genetic diversity. Some site features seem to affect the clonal structure at the local scale, influencing the relocation of detached fragments and the level of intermingling, but they do not substantially affect genetic diversity. The peculiar vegetative reproduction coupled with somatic mutation could partly account for the genetic variation detected. Genetic distances highlight geographic isolation and limited gene flow among populations. We found only two trnL haplotypes in Europe due to length polymorphism, but, over an intercontinental scale, only non-delete trnL was found in Africa and the USA. ISSR analysis within each population detected a higher genetic distance between the samples with different trnL haplotypes, suggesting the presence of two different genetic lineages within this species, geographically overlapping in the Mediterranean Basin.