A polyphasic molecular approach was used in order to characterize and taxonomically assign Debaryomyces yeast isolates of different origins. Actin 1 (ACT1) gene sequences coupled with AFLP markers showed that the investigated yeasts belonged to the recently reinstated species D. hansenii, D. fabryi and D. tyrocola. The strain HA1179 was supposed to be a D. hansenii strain with introgressed D. fabryi DNA segments. This strain acquired ribosomal RNA encoding genes (rDNA) and the ACT1 gene from the species D. fabryi and D. hansenii respectively. Comparative sequence analysis of the ACT1 gene, ITS1-5.8S-ITS2 (5.8S-ITSs) and D1/D2 regions, suggested that five strains isolated from a municipal wastewater treatment plant could represent a new taxon of the genus, for which the name Debaryomyces vindobonensis was proposed. The calculated degree of similarity between the AFLP patterns indicated that the strains of D. vindobonensis and the closely related species were separated by the values ＜0.5. New yeast isolates showed very similar morphological and physiological properties to related Debaryomyces species. They differed notably only by the assimilation of rhamnose and growth at 50% glucose. In contrast to the other species, D. vindobonensis was unable to assimilate starch.