AbstractMutations that add, subtract, rearrange, or otherwise refashion genome structure often affect phenotypes, although the fragmented nature of most contemporary assemblies obscures them. To discover such mutations, we assembled the first new reference-quality genome ofDrosophila melanogastersince its initial sequencing. By comparing this new genome to the existingD. melanogasterassembly, we created a structural variant map of unprecedented resolution and identified extensive genetic variation that has remained hidden until now. Many of these variants constitute candidates underlying phenotypic variation, including tandem duplications and a transposable element insertion that amplifies the expression of detoxification-related genes associated with nicotine resistance. The abundance of important genetic variation that still evades discovery highlights how crucial high-quality reference genomes are to deciphering phenotypes.