Wolbachia are among the most widespread symbionts on the earth. They spread within populations by various means of manipulating host reproduction, including cytoplasmic incompatibility (CI), male-killing (MK), parthenogenesis, and feminization. Phylogenetic analyses indicate that Wolbachia have the potential to undergo rapid evolutionary change in phenotype, for example, from CI to MK, although such analyses do not reveal the rate at which such transitions occur, nor the nature of the intermediate phenotypes. Here I show that a transition from CI to MK can occur almost instantaneously on an evolutionary time scale. A Wolbachia strain that causes CI in its natural host, Drosophila recens, was introgressed to its sister species D. subquinaria via the natural processes of hybridization and backcrossing. In some strains of D. subquinaria, infection with this Wolbachia strain caused essentially complete MK, resulting in all-female broods, whereas in other strains, there was no effect on offspring sex ratio. Crosses within and between D. subquinaria and D. recens revealed that resistance to MK is dominant, autosomal, multigenic, and dependent on zygotic, not maternal, genotype. MK in D. subquinaria is unusual in that the male offspring of infected females die during the larval stage, not as embryos. These findings suggest that MK and CI may share a similar underlying molecular basis.