The brown bear (Ursus arctos) is the largest terrestrial carnivore that inhabits the Mountain District of Gorski kotar and the geographical area of Lika. It is included in the red list of the endangered species in Croatia. Malassezia pachydermatis yeast, which is considered an opportunistic pathogen, causes various pathological skin disorders in pets under certain predisposing conditions, as well as in domestic and wild animals. There is very little data about the presence and role of M. pachydermatis on the bear skin. The first aim of the study was to optimize a genomic DNA extraction protocol from the M. pachydermatis culture growing on two different culture media. The specific aim was the amplification of particular DNA fragments using ITS1 and LSU primer sequences in order to describe the genetic variation of Malassezia pachydermatis yeast in the brown bear (Ursus arctos) on the territory of Croatia. The frozen archived yeast strains were grown on two different culture media, the genomic DNA was isolated using a commercially available kit and the specific DNA fragments (ITS1 and LSU regions) were amplified by a polymerase chain reaction. In addition, the nucleotide sequences were analyzed. The results have shown that the isolated yeast colonize both the epithelium of the ear canal and the perianal area of the brown bear. The multiplied DNA fragments correspond to the previously published length of the ITS1 and LSU regions. It has been noticed that the analyzed regions contain certain varieties of the nucleotide sequence and that the LSU region is much better preserved than the ITS1 region. These preliminary results indicate possible evolutionary preservation, which needs to be further explored by analyzing a larger number of samples and by introducing additional molecular research methods.