AbstractAlthough single genes underlying several evolutionary adaptations have been identified, the genetic basis of complex, polygenic adaptations has been far more challenging to pinpoint. Here we report that the budding yeastSaccharomyces paradoxushas recently evolved resistance to citrinin, a naturally occurring mycotoxin. Applying a genome-wide test for selection oncis-regulation, we identified five genes involved in the citrinin response that are constitutively up-regulated inS. paradoxus.Four of these genes are necessary for resistance, and are also sufficient to increase the resistance of a sensitive strain when over-expressed. Moreover,cis-regulatory divergence in the promoters of these genes contributes to resistance, while exacting a cost in the absence of citrinin. Our results demonstrate how the subtle effects of individual regulatory elements can be combined, via natural selection, into a complex adaptation. Our approach can be applied to dissect the genetic basis of polygenic adaptations in a wide range of species.Author SummaryAdaptation via natural selection has been a subject of great interest for well over a century, yet we still have little understanding of its molecular basis. What are the genetic changes that are actually being selected? While single genes underlying several adaptations have been identified, the genetic basis of complex, polygenic adaptations has been far more challenging to pinpoint. The complex trait that we study here is the resistance ofSaccharomycesyeast to a mycotoxin called citrinin, which is produced by many other species of fungi, and is a common food contaminant for both humans and livestock. We found that sequence changes in the promoters of at least three genes have contributed to citrinin resistance, by up-regulating their transcription even in the absence of citrinin. Higher expression of these genes confers a fitness advantage in the presence of citrinin, while exacting a cost in its absence— a fitness tradeoff. Our results provide a detailed view of a complex adaptation, and our approach can be applied to polygenic adaptations in a wide range of species.