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The significance of prezygotic isolation for speciation has been recognized at least since the Modern Synthesis. However, fundamental questions remain. For example, how are genetic associations between traits that contribute to prezygotic isolation maintained? What is the source of genetic variation underlying the evolution of these traits? And how do prezygotic barriers affect patterns of gene flow? We address these questions by reviewing genetic features shared across plants and animals that influence prezygotic isolation. Emerging technologies increasingly enable the identification and functional characterization of the genes involved, allowing us to test established theoretical expectations. Embedding these genes in their developmental context will allow further predictions about what constrains the evolution of prezygotic isolation. Ongoing improvements in statistical and computational tools will reveal how pre- and postzygotic isolation may differ in how they influence gene flow across the genome. Finally, we highlight opportunities for progress by combining theory with appropriate data.