1 CECT (Spanish Type Culture Collection), Universitat de València, Edificio de Investigación, Campus de Burjassot, 46100 Burjassot, Valéncia, Spain 2 Departamento de Biotecnologia, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), P. Box 73, 46100 Burjassot, València, Spain Author for correspondence: A. Querol. Tel: +34 6 390 00 22. Fax: +34 6 363 63 01. e-mail: aquerol{at}iata.csic.es ABSTRACT The identification and classification of yeasts have traditionally been based morphological, physiological and biochemical traits. Various kits have been developed as rapid systems for yeast identification, but mostly for clinical diagnosis. In recent years, different molecular biology techniques have been developed for yeast identification, but there is no available database to identify a large number of species. In the present study, the restriction patterns generated from the region spanning the internal transcribed spacer (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts. In many cases, the size of the PCR products and the restriction patterns obtained with endonucleases C fol, Hae lll and Hin fl yielded a unique profile for each species. Accordingly, the use of this molecular approach is proposed as a new rapid and easy method of routine yeast identification. Key Words: yeast identification • RFLP • 5.8S-ITS region