The fungus Pyrenophora graminea is the causal agent of barley leaf stripe disease. Two leaf stripe isolates PgSy3 (exhibiting high virulence on the barley cultivar ``Arabi Abiad``) and PgSy1 (exhibiting low virulence on Arabi Abiad), were mated and 63 progeny were isolated and phenotyped for the reaction on Arabi Abiad. The population segregated in a 1:1 ratio, 32 virulent to 31 avirulent (χ2 = 0.05, P = 0.36), indicating single gene control of PgSy3 virulence on Arabi Abiad. Among 96 AFLP markers identified, three AFLP markers, E37M50-400, E35M59-100 and E38M47-800 were linked to the virulence locus VHv1 in isolate PgSy3. The results of this study indicate that (the three markers) are closely linked to VHv1 and are unique to isolates carrying the virulence locus. This work represents an initial step towards map-based cloning of VHv1 in P. graminea.