The genetic structure of Pyrenophora teres, an ascomycete fungus that causes net blotch of barley, was examined using random amplified polymorphic DNA (RAPD) markers. Twenty-seven random oligonucleotide primers were screened against DNA from 16 isolates of P. teres of diverse geographic origin. Five primers gave scorable, reproducible DNA products (bands) suitable for population genetic studies. Genetic analyses of bands produced by two of the primers revealed single locus segregation in three of four crosses, indicating that these RAPDs can be interpreted as alleles at genetic loci. Allele frequencies were determined for 10 putative RAPD loci from five primers in 22-35 isolates of P. teres sampled from each of five geographically separated populations in Canada, Germany, and the U.S.A. Eight RAPD loci were polymorphic in at least one population and two loci were monomorphic in all five populations. Variation in allele frequencies (allelic diversity) among the five populations was partitioned into within- and among-population components using Nei's G(ST). A G(ST) value of 0.46 was obtained among all populations indicating that approximately 46% of the total genetic variability detected was due to differentation among populations compared with 54% within populations. A G(ST) value of 0.33 was obtained among the North American populations only. From five to nine multilocus genotypes were found in each population. Nine genotypes occurred exclusively in the German population and four exclusively in the New York population. The other populations had one or two unique genotypes. Gametic disequilibrium values (nonrandom associations of RAPD loci) were calculated among all pairs of polymorphic loci within each population. Eleven of 49 values were significantly different from 0 (P < 0.05); 8 of the 11 significant gametic disequilibrium values occurred in the New York population. Highly significant gametic disequilibrium was detected between the same two RAPD loci in three different populations, suggesting that these loci are genetically linked. Two different multilocus analyses revealed that the genetic structures of the Alberta, North Dakota, and German populations but not the New York population were consistent with random sexual reproduction occurring in these populations.