The pathogenicity to wheat (Pwt1) locus conditions host species specificity of Magnaporthe oryzae on wheat. GFSI1-7-2 (Setaria isolate) carries the avirulence allele (PWT1) at this locus while Br48 (Triticum isolate) carries the virulence allele (pwt1). An F(1) culture derived from a cross between GFSI1-7-2 and Br48 was backcrossed with Br48 to produce a tester population in which PWT1 alone segregated. When hexaploid wheat lines were inoculated with the BC(1)F(1) testers, they were all resistant to all PWT1 carriers and susceptible to all pwt1 carriers, suggesting that they recognize PWT1. When barley cultivars were inoculated with the testers, they showed the same pattern of reactions as the hexaploid lines, suggesting that the barley cultivars also recognize PWT1. These results suggest that PWT1 is a fundamental gene that universally conditions the avirulence of Setaria isolates on two staple crops, hexaploid wheat and barley. Interestingly, tetraploid wheat lines did not recognize PWT1. Molecular mapping using the F(1) and BC(1)F(1) populations revealed that the Pwt1 locus is located on chromosome 2 and tightly linked to the ribosomal DNA locus and a telomere.