SUMMARY One of the primary goals of genetics is to understand how and why naturally varying individuals differ in phenotype. Mapping the molecular basis of this trait variation can be straightforward, but only in individuals that can be interbred. We have found that a little-studied worm species, Caenorhabditis latens, has a median lifespan 50% longer than related nematodes, and we have observed robust, conserved thermotolerance in Saccharomyces cerevisiae, the only species of its clade that can act as an opportunistic pathogen. In the current proposal, we dissect these species divergences using a massively parallel version of the reciprocal hemizygote test. We create a genomic complement of hemizygote mutants, by generating viable, sterile F1 hybrids between species and subjecting them to transposon mutagenesis. We pool the hemizygotes, measure their longevity and fitness in sequencing-based assays, and test for differences in frequency between clones of the pool bearing the two parents' alleles of a given gene. The result is a catalog of loci at which variants between species influence the trait of interest. In our proof of principle using yeast (Aim 1) and worm (Aim 2), we will uncover alleles that have arisen in wild species to boost healthspan and stress resistance. These results will stand in contrast to the alleles weakening fitness that are often mapped in intra-specific studies. Orthologs of our yeast loci will be of immediate interest as candidates for virulence genes in prevalent fungal pathogens, and orthologs of our worm lifespan factors will be well-suited to analysis for anti-aging effects in mammals. This effort will be the first-ever comprehensive survey of the genetic architecture and molecular genetics of trait variation between reproductively isolated individuals. And the methods we pioneer will enable genetic dissection in any eukaryotic system in which banks of F1 hybrid individuals or their tissues are available.