PCR primers were developed for the specific detection of Clavispora lusitaniae, Debaryomyces hansenii var hansenii, Geotrichum candidum, Kluyveromyces lactis and K. marxianus and Yarrowia lipolytica, yeast species commonly found on the surface of smear cheese. Forty eight representative strains frequently found in smear cheeses or taxonomically related to the target yeasts were used as templates, to validate the designed primers. The specific and selective detection of these yeasts was effective in situ, in Livarot smear, without yeast isolation and culture and was comparable with data obtained with a conventional method. The primers described here have thus potential for PCR studies applied to cheese. It should also be possible to use some of these primers with other substrates.