The genusWolbachiais an archetype of maternally inherited intracellular bacteria that infect the germline of numerous invertebrate species worldwide. They can selfishly alter arthropod sex ratios and reproductive strategies to increase the proportion of the infected matriline in the population. The most common reproductive manipulation is cytoplasmic incompatibility, which results in embryonic lethality in crosses between infected males and uninfected females. Females infected with the sameWolbachiastrain rescue this lethality. Despite more than 40 years of research1and relevance to symbiont-induced speciation2,3, as well as control of arbovirus vectors4,5,6and agricultural pests7, the bacterial genes underlying cytoplasmic incompatibility remain unknown. Here we use comparative and transgenic approaches to demonstrate that two differentially transcribed, co-diverging genes in the eukaryotic association module of prophage WO8fromWolbachiastrainwMel recapitulate and enhance cytoplasmic incompatibility. Dual expression in transgenic, uninfected males ofDrosophila melanogastercrossed to uninfected females causes embryonic lethality. Each gene additively augments embryonic lethality in crosses between infected males and uninfected females. Lethality associates with embryonic defects that parallel those of wild-type cytoplasmic incompatibility and is notably rescued bywMel-infected embryos in all cases. The discovery of cytoplasmic incompatibility factor genescifAandcifBpioneers genetic studies of prophage WO-induced reproductive manipulations and informs the continuing use ofWolbachiato control dengue and Zika virus transmission to humans.