Pyrenophora teres f. sp. teres mating-type genes (MAT-1: 1190 bp; MAT-2: 1055 bp) have been identified. Their predicted proteins, measuring 379 and 333 amino acids, respectively, are similar to those of other Pleosporales, such as Pleospora sp., Cochliobolus sp., Alternaria alternata, Leptosphaeria maculans, and Phaeosphaeria nodorum. The structure of the MAT locus is discussed in comparison with those of other fungi. A mating-type PCR assay has also been developed; with this assay we have analyzed 150 isolates that were collected from 6 Sardinian barley landrace populations. Of these, 68 were P. teres f. sp. teres (net form; NF) and 82 were P. teres f. sp. maculata (spot form; SF). Within each mating type, the NF and SF amplification products were of the same length and were highly similar in sequence. The 2 mating types were present in both the NF and the SF populations at the field level, indicating that they have all maintained the potential for sexual reproduction. Despite the 2 forms being sympatric in 5 fields, no intermediate isolates were detected with amplified fragment length polymorphism (AFLP) analysis. These results suggest that the 2 forms are genetically isolated under the field conditions. In all of the samples of P. teres, the ratio of the 2 mating types was consistently in accord with the 1:1 null hypothesis. This ratio is expected when segregation distortion and clonal selection among mating types are absent or asexual reproduction is rare. Overall, sexual reproduction appears to be the major process that equalizes the frequencies of the 2 mating types within populations.