Several features of the yeast mitochondrial genome, including high mutation rate, dynamic genomic structure, small effective population size, and dispensability for cellular viability, make it a promising candidate for generating hybrid incompatibility and driving speciation. Cytonuclear incompatibility, a specific type of Dobzhansky-Muller genetic incompatibility caused by improper interactions between mitochondrial and nuclear genomes, has previously been observed in a variety of organisms, yet its role in speciation remains obscure. Recent studies in Saccharomyces yeast species provide a new insight, with experimental evidence that cytonuclear incompatibility and DNA sequence divergence are both causes of the reproductive isolation of different yeast species. Interestingly, these two mechanisms seem to be perfectly complementary to each other in terms of their effects and evolutionary trajectories. Direct molecular analyses of the incompatible genes in yeasts have started to shed light on the evolutionary forces driving speciation.