In the laboratory, the ascomycete fungus Cryphonectria parasitica is rarely self-fertile, and has a self-incompatibility system that resolves into two intersterility groups, controlled by a single locus. In natural populations, however, self-fertilization occurs frequently. In this report, we show that the C. parasitica self-incompatibility locus (MAT) comprises two idiomorphs (alleles that are highly divergent in sequence), conforming to the paradigm of self-incompatibility as described for other ascomycetes. Starting with a fragment putatively from the MAT-2 idiomorph, we used a PCR-based cloning approach to identify 3.5- and 2-kb sequences unique to MAT-1 and MAT-2 isolates, respectively. These sequences were then used to design idiomorph-specific PCR primer pairs, allowing us to efficiently identify the mating types of isolates, a crucial component of our research on the environmental and genetic factors underlying this mixed mating system.