Theoretical models have shown that speciation with gene flow can occur readily via a "one-allele mechanism," where the spread of the same allele within both of two diverging species reduces their subsequent hybridization. Here we present direct genetic evidence for such an allele in Drosophila pseudoobscura. Alleles conferring high or low assortative mating in D. pseudoobscura produce the same effects when inserted into D. persimilis. This observation suggests that the type of genetic variation that is most conducive to controversial modes of speciation with gene flow, such as reinforcement or sympatric speciation, is present in nature.