Clinically relevant features of monogenic diseases, including severity of symptoms and age of onset, can vary widely in response to environmental differences as well as to the presence of genetic modifiers affecting the trait’s penetrance and expressivity. While a better understanding of modifier loci could lead to treatments for Mendelian diseases, the rarity of individuals harboring both a disease causing allele and a modifying genotype hinders their study in human populations. Our interest in studying the effect of modifiers on monogenic traits has led us to investigate a dimorphic trait of Saccharomyces cerevisiae when grown on a colorimetric medium used in clinical identification of fungal pathogens. While the majority of natural isolates of S. cerevisiae develop a distinct purple color on this medium, a small number remain white, demonstrating that this trait is dimorphic in the population. To characterize the nature of this variation further, we preformed QTL analysis on a large number of recombinant progeny produced by a cross between white and purple parental strains. Our results identified a major QTL peak over a region that includes the tandemly arrayed paralogs PHO3 and PHO5, suggesting the colorimetric assay is an indicator of acid phosphatase activity. Sanger sequencing of these loci in 28 white strains in our natural variant collection revealed most strains harbored a gene fusion of the two phosphatase genes that deleted the constitutively expressed PHO3 promoter region. Gradations in color among the progeny of the cross suggested additional genes, unlinked to the PHO3- PHO5 locus, could modify the acid phosphatase activity. We used time-lapse photography to quantitatively measure the growth and development of purple color of each individual colony over time. By conditioning on the major QTL allele, we identified additional secondary QTL peaks that differed between the two groups and observed a QTL peak with a LOD score that declined over the time course of the experiment. In order to identify modifiers of acid phosphatase activity, we are currently applying a novel high throughput method to isolate informative recombinant progeny for targeted genotyping.