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The reciprocal hemizygosity test is a straightforward genetic test that can positively identify genes that have evolved to contribute to a phenotypic difference between strains or between species. The test involves a comparison between hybrids that are genetically identical throughout the genome except at the test locus, which is rendered hemizygous for alternative alleles from the two parental strains. If the two reciprocal hemizygotes display different phenotypes, then the two parental alleles must have evolved. New methods for targeted mutagenesis will allow application of the reciprocal hemizygosity test in many organisms. This review discusses the principles, advantages, and limitations of the test.