The budding yeast Saccharomyces cerevisiae is the best studied eukaryote in molecular and cell biology, but its utility for understanding the genetic basis of natural phenotypic variation is limited by the inefficiency of association mapping owing to strong and complex population structure. To facilitate association mapping, we analyzed 190 high-quality genomes of diverse strains, including 85 newly sequenced ones, to uncover yeast's population structure that varies substantially among genomic regions. We identified 181 yeast genes that are absent from the reference genome and demonstrated their expression and role in important functions such as drug resistance. We then simultaneously measured the growth rates of over 4500 lab strains each deficient of a nonessential gene and 81 natural strains across multiple environments using unique DNA barcode present in each strain. We combined the genome-wide reverse genetic information with genome-wide association analysis to determine potential genomic regions of importance to environmental adaptations, and for a subset experimentally validated their role by reciprocal hemizygosity tests. The resources provided permit efficient and reliable association mapping in yeast and significantly enhances its value as a model for understanding the genetic mechanisms of phenotypic polymorphism and evolution.