Antifungal drug resistance in the pathogenic fungus Candida albicans is a serious threat to the growing population of immunocompromised patients. This study describes a significant correlation between loss of heterozygosity at the C. albicans mating-type-like (MTL) locus and resistance to azole antifungals. A pool of 96 clinical isolates consisting of 50 azole-resistant or susceptible dose-dependent isolates and 46 azole-susceptible isolates was screened by PCR for the presence of MTLa1 and MTLalpha1. These genes were used as markers for the MTLa and MTLalpha loci. Both loci were present in 84 of the isolates. Six isolates failed to amplify MTLa1 and six failed to amplify MTLalpha1. Further PCR analysis demonstrated that loss of the MTLa1 and MTLalpha1 genes corresponded to loss of all of the loci-specific genes, resulting in homozygosity at the MTL locus. Southern analysis and single nucleotide polymorphism (SNP) analysis were used to determine that this loss of heterogeneity was due to replacement of one of the MTL loci with a duplicate of the other locus resulting in two homozygous copies of the MTL locus. Of the 12 homozygous isolates, one isolate was sensitive to azole drugs. Statistical analysis of the data demonstrates a strong correlation between homozygosity at the MTL locus and azole resistance (P<0 small middle dot003). In a set of serial isolates, an increase in azole resistance correlated with the loss of heterozygosity at the MTL locus, lending further strength to the correlation. Gene disruptions of the MTL loci were found to have no effect on azole susceptibility.