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Candida albicans is the most prevalent opportunistic fungal pathogen in the clinical setting, causing a wide spectrum of diseases ranging from superficial mucosal lesions to life-threatening deep-tissue infections. Recent studies provide strong evidence that C. albicans possesses an arsenal of genetic mechanisms promoting genome plasticity and that it uses these mechanisms under conditions of nutritional or antifungal drug stress. Two microarray-based methods, single nucleotide polymorphism (SNP) and comparative genome hybridization arrays, have been developed to study genome changes in C. albicans. However, array technologies can be relatively expensive and are not available to every laboratory. In addition, they often generate more data than needed to analyze specific genomic loci or regions. Here, we have developed a set of SNP-restriction fragment length polymorphism (RFLP) (or PCR-RFLP) markers, two per chromosome arm, for C. albicans. These markers can be used to rapidly and accurately detect large-scale changes in the C. albicans genome including loss of heterozygosity (LOH) at single loci, across chromosome arms or across whole chromosomes. Furthermore, skewed SNP-RFLP allelic ratios are indicative of trisomy at heterozygous loci. While less comprehensive than array-based approaches, we propose SNP-RFLP as an inexpensive, rapid, and reliable method to screen strains of interest for possible genome changes. © 2009 Federation of European Microbiological Societies.