Pyrenophora graminea, the causal agent of leaf stripe disease, is an economically important pathogen of barley found worldwide. Forty-four isolates of diverse geographical origin within Syria were grouped into vegetative compatibility groups (VCGs) by demonstrating heterokaryosis by complementation tests using nitrate nonutilizing (nit) mutants. All isolates were grouped into three VCGs-1-A, 1-B and 1-C. No self-incompatibility was observed in any of the isolates tested. VCG 1-A was the most common group within growing regions in Syria and proved to be the most virulent of the VCGs identified. These data indicate that the level of virulence in P. graminea is related to VCG.