A Pyrenophora teres f. teres cross between isolates 0-1 and 15A was used to evaluate the genetics of avirulence associated with barley lines Canadian Lake Shore (CLS), Tifang, and Prato. 15A is avirulent on Tifang and CLS, but virulent on Prato. Conversely, 0-1 is avirulent on Prato, but virulent on Tifang and CLS. Avirulence:virulence on Tifang and CLS segregated 1:1, whereas avirulence:virulence on Prato segregated 3:1. An AFLP-based linkage map was constructed and used to identify a single locus derived from 15A (AvrHar) conferring avirulence to Tifang and CLS. Virulence on Prato was conferred by two epistatic genes (AvrPra1 and AvrPra2). AvrPra2 co-segregated with AvrHar, but the two genes from opposite parents conferred opposite reactions. This work provides the foundation for the isolation of these avirulence genes.