In the opportunistic fungal pathogen Candida albicans, up-regulation of MDR1, encoding an efflux transporter, leads to increased resistance to the antifungal drug fluconazole. Antifungal resistance has been linked to several types of genetic change in C. albicans, including changes in genome structure, genetic alteration of the drug target, and overexpression of transporters. High-level over-expression of MDR1 is commonly mediated by mutation in a trans-acting factor, Mrr1p. This report describes a second mechanism that contributes to up-regulation of MDR1 expression. By analyzing the sequence of the MDR1 promoter region in fluconazole-resistant and fluconazole-susceptible strains, we identified sequence polymorphisms that defined two linkage groups, corresponding to the two alleles in the diploid genome. One of the alleles conferred higher MDR1 expression compared with the other allele. Strains in which both alleles were of the higher activity type were common in collections of clinically isolated strains while strains carrying only the less active allele were rare. As increased expression of MDR1 confers higher resistance to drugs, strains with the more active MDR1 promoter allele may grow or survive longer when exposed to drugs or other selective pressures, providing greater opportunity for mutations that confer high-level drug resistance to arise. Through this mechanism, higher activity alleles of the MDR1 promoter could promote the development of drug resistance.