Although prezygotic isolation between sympatric populations of closely related animal and plant species is well documented, far less is known about such evolutionary phenomena in sexual microbial species, as most are difficult to culture and manipulate. Using the molecular and genetic tools available for the unicellular fungus Saccharomyces cerevisiae, and applying them to S. paradoxus, we tested the behavior of individual cells from sympatric woodland populations of both species for evidence of prezygotic isolation. First, we confirmed previous observations that vegetative cells of both species mate preferentially with S. cerevisiae. Next, we found evidence for mate discrimination in spores, the stage in which outcrossing opportunities are most likely to occur. There were significant differences in germination timing between the species: under the same conditions, S. paradoxus spores do not begin germinating until almost all S. cerevisiae spores have finished. When germination time was staggered, neither species discriminated against the other, suggesting that germination timing is responsible for the observed mate discrimination. Our results indicate that the mechanisms of allochronic isolation that are well known in plants and animals can also operate in sexual microbes.