ABSTRACT Permanent changes in gene expression result from certain forms of antifungal resistance. In this study, we asked whether any changes in gene expression are required for the evolution of a drug-resistant phenotype in populations. We examined the changes in gene expression resulting from the evolution of resistance in experimental populations of the yeast Saccharomyces cerevisiae with two antifungal drugs, fluconazole (FLC) in a previous study and amphotericin B (AmB) in this study, in which five populations were subjected to increasing concentrations of AmB, from 0.25 to 128 μg/ml in twofold increments. Six genes, YGR035C, YOR1 , ICT1 , GRE2 , PDR16 , and YPLO88W, were consistently overexpressed with resistance to AmB reported here and with resistance to FLC involving a mechanism of increased efflux reported previously. We then asked if the deletion of these genes impaired the ability of populations to evolve resistance to FLC over 108 generations of asexual reproduction in 32 and 128 μg/ml FLC, the same conditions under which FLC-resistant types evolved originally. For each of three deletion strains, YOR1 , ICT1 , and PDR16 strains, extinctions occurred in one of two replicate populations growing in 128 μg/ml FLC. Each of these three deletion strains was mixed 1:1 with a marked version of the wild type to measure the relative ability of the deletion strain to adapt over 108 generations. In these assays, only the PDR16 deletion strain consistently became extinct both at 32 and at 128 μg/ml FLC. The deletion of PDR16 reduces the capacity of a population to evolve to resistance to FLC.