Over the last few decades the emergence of resistance to commonly used antifungal molecules has become a major barrier to effective treatment of recurrent life threatening fungal diseases. This together with the increase incidence of fungal diseases has created the need for new antifungals with different mechanisms of action to broaden treatment options. Antimicrobial peptides produced in plants and animals are promising new molecules in the arsenal of antifungal agents because they satisfy the recruitment for different mechanisms of action and are often targeted specifically to fungal pathogens (van der Weerden, Bleackley et al., 2013). A key step in the development of novel antifungals is understanding the potential for the fungus to develop resistance. Here we have used the prototypic plant defensin NaD1 in serial passages with the model fungus S. cerevisiae to examine evolution of resistance to this class of antifungal. The yeast strains did develop tolerance to NaD1 more slowly than to the clinically used antifungal caspofungin. Sequencing the genomes of the strains with increased tolerance failed to identify any hotspot mutations associated with increased tolerance and found 12 genes that are involved in resistance. Characterization of the strains with increased tolerance to NaD1 also revealed changes in tolerance to a number of abiotic stressors. Resistance developed slowly via an accumulation of single nucleotide mutations, and had a fitness penalty associated with it. One of the genes identified FPS1, indicates that there is a common mechanism of resistance to NaD1 involving the osmotic stress response pathway. This data indicates that resistance to antimicrobial peptides such as NaD1 is more difficult to achieve when compared to small molecule antifungals.