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The genetic diversity of two major yeast species found in cheese, Debaryomyces hansenii and Kluyveromyces marxianus, was analyzed within the yeast flora in French traditional cheesemaking. Based on the amplification of sequences separating long terminal repeat (LTR) retrotransposon sequences, a molecular typing method was developed for D. hansenii and K. marxianus. This method was applied to a total of 56 D. hansenii strains and 61 K. marxianus strains, mostly isolated during fermentation and ripening of traditional cheese from Normandy and Haute-Savoie (French Alps) regions. A total of 32 and 43 robust profiles were obtained for D. hansenii and K. marxianus, respectively. Cluster analysis confirmed the large genetic diversity already shown for D. hansenii and revealed an even larger diversity for K. marxianus. After its use with Saccharomyces cerevisiae, the inter-LTR PCR proved to be efficient to discriminate between strains of the two species, D. hansenii and K. marxianus, isolated from the same ecological niches, confirming the high intra-specific variability of species found in cheese. This strain typing could not correlate the analyzed strains with their origin, would it be the cheese type, the cheese-making facility or the cheese batch, showing a high discrimination power. The method described here will provide a fast and reliable tool for the biodiversity study of these two major cheese yeasts.