Speciation of fungal plant pathogens has been associated with host jumps, host domestication, clonal divergence, and hybridization. Although we have substantial insight into the speciation histories of several important plant pathogens, we still know very little about the underlying genetics of reproductive isolation. Studies in Saccharomyces cerevisiae, Neurospora crassa, and nonfungal model systems illustrate that reproductive barriers can evolve by different mechanisms, including genetic incompatibilities between neutral and adaptive substitutions, reinforcement selection, and chromosomal rearrangements. Advances in genome sequencing and sequence analyses provide a new framework to identify those traits that have driven the divergence of populations or caused reproductive isolation between species of fungal plant pathogens. These traits can be recognized based on signatures of strong divergent selection between species or through the association of allelic combination conferring hybrid inferiority. Comparative genome analyses also provide information about the contribution of genome rearrangements to speciation. This is particularly relevant for species of fungal pathogens with extreme levels of genomic rearrangements and within-species genome plasticity.