Complex traits typically involve the contribution of multiple gene variants. In this study, we took advantage of a high-density genotyping analysis of the BY (S288c) and RM strains of Saccharomyces cerevisiae and of 123 derived spore progeny to identify the genetic loci that underlie a complex DNA repair sensitivity phenotype. This was accomplished by screening hybrid yeast progeny for sensitivity to a variety of DNA damaging agents. Both the BY and RM strains are resistant to the ultraviolet light-mimetic agent 4-nitroquinoline 1-oxide (4-NQO); however, hybrid progeny from a BYxRM cross displayed varying sensitivities to the drug. We mapped a major quantitative trait locus (QTL), RAD5, and identified the exact polymorphism within this locus responsible for 4-NQO sensitivity. By using a backcrossing strategy along with array-assisted bulk segregant analysis, we identified one other locus, MKT1, and a QTL on Chromosome VII that also link to the hybrid 4-NQO-sensitive phenotype but confer more minor effects. This work suggests an additive model for sensitivity to 4-NQO and provides a strategy for mapping both major and minor QTL that confer background-specific phenotypes. It also provides tools for understanding the effect of genetic background on sensitivity to genotoxic agents.