Individual response to small-molecule drugs is variable; a drug that provides a cure for some may confer no therapeutic benefit or trigger an adverse reaction in others. To begin to understand such differences systematically, we treated 104 genotyped segregants from a cross between two yeast strains with a collection of 100 diverse small molecules. We used linkage analysis to identify 124 distinct linkages between genetic markers and response to 83 compounds. The linked markers clustered at eight genomic locations, or quantitative-trait locus 'hotspots', that contain one or more polymorphisms that affect response to multiple small molecules. We also experimentally verified that a deficiency in leucine biosynthesis caused by a deletion of LEU2 underlies sensitivity to niguldipine, which is structurally related to therapeutic calcium channel blockers, and that a natural coding-region polymorphism in the inorganic phosphate transporter PHO84 underlies sensitivity to two polychlorinated phenols that uncouple oxidative phosphorylation. Our results provide a step toward a systematic understanding of small-molecule drug action in genetically distinct individuals.