We developed a rapid and sensitive identification method for the halotolerant yeast Debaryomyces hansenii, based on the hybridization of species-specific sequences. These sequences were first identified in a survey of D. hansenii strains by random amplification of polymorphic DNA (RAPD) as giving conserved bands in all isolates tested. Two such conserved RAPD products, termed F01pro and M18pro, were cloned from the type strain CBS 767. The specificity of these probes was assessed by hybridizing them to DNA from various species of yeasts commonly found in cheese. F01pro and M18pro hybridized to the DNA of all D. hansenii var. hansenii tested, but not to DNA of other yeast species including the closely related strain of D. hansenii var. fabryii CBS 789. Hybridization patterns of F01pro and M18pro on digested genomic DNA of D. hansenii indicated that the sequences were repeated in the genome of all D. hansenii var. hansenii tested, and gave distinct polymorphic patterns. The single F01pro probe generated 11 different profiles for 24 strains by restriction fragment length polymorphism, using one restriction enzyme. F01pro represents a new type of repeated element found in fungi, useful for both identification and typing of D. hansenii and, together with M18pro, simplifies the study of this species in complex flora. ß