The origin of a new species requires a mechanism to prevent divergent populations from interbreeding. In the classic allopatric model, divided populations evolve independently and accumulate genetic differences. If contact is restored, hybrids suffer reduced fitness and selection may favor traits that prevent mistakes in mating, a process known as reinforcement. This decisive but transient phase is challenging to document and has been reported mostly in macroorganisms. Very little is known about the processes through which new microbial species originate. In particular, it is unclear whether microbial eukaryotes, many of which can reproduce sexually during complex life cycles, speciate in much the same way as do well-studied plants and animals. Using individual cellular mate choice trials, we investigated the mating behavior of sympatric and allopatric woodland populations of the yeast Saccharomyces paradoxus. We find evidence consistent with reinforcement, potentially representing an example of microbial speciation in progress.