Candida rugosa is a rare as yet, though emerging pathogen, attracting increasing international interest. A collection of 10 clinical strains from the southern Balkan peninsula was subjected to polyphasic detection, identification and typing techniques, such as cultures in differential media, microscopy, assimilation tests, immunefluorescence, in vitro drug susceptibility (microdilution CLSI methodology and E-test) to multiple agents and both simple and amplification-based molecular techniques, such as electrophoretic karyotyping, RFLP-PFGE, PCR fingerprinting with mini- and microsatellite DNA, ribotyping and multiple PCR-RFLPs. Similarities and differences to other members of the genus were noted to determine appropriate identification, typing and treatment techniques and regimens for use in epidemiological studies and clinical applications, as the resulting C. rugosa profiles were in different degrees distinct but generally not dissimilar to other Candida species. TTC assimilation and Dalmau cultures allow identification, but typing is better achieved with electrophoretic karyotyping and specific PCR-RFLP, though the respective results are similar but not matching, indicating discontinuous intraspecific microevolutionary events.