The genetic structure of 19 populations of two species of the genus Ptilidium – P. pulcherrimum and P. ciliare from Poland and Slovakia was studied. Nine putative loci in nine enzyme systems were resolved. Two loci (Got and Me) were monomorphic at the genus level. Both species were fixed for alternative alleles at three loci (Acp, Idh and Prx- A) that can be used as diagnostic markers for these two species. Both species proved to be polymorphic: P. ciliare for three (Gdh, Pgm and Sdh), and P. pulcherrimum for four loci (Gdh, Pgm, Sdh and Pgd). Gene diversity statistics show a lower total gene diversity (HT) of asexually reproducing P. ciliare (0.299 ± 0.089) as compared to sexually reproducing P. pulcherrimum (0.379 ± 0.044). The GST in P. ciliare was 0.263, in P. pulcherrimum 0.374. In both species a relation between the allele numbers and allele frequencies with geographical regions was observed. Montane populations of P. ciliare displayed a lower number of alleles than the populations derived from the lowlands, whereas in P. pulcherrimum no genetic differentiation between both geographical regions was observed.