Cochliobolus sativus, the causal agent of barley spot blotch disease, is able to undergo spontaneous, vegetative cell fusion. In this study, the utility of enzymatic mismatch cleavage for discovery of single-nucleotide variations in Syrian Vegetative Compatibility Groups (VCGs) obtained from pairing complementary nit mutants of C. sativus isolates was investigated for the first time. Gene-specific primers were designed from the whole fungal genome for use in the Ecotilling assays on VCGs isolates. Differences of band patterns among the digested products of different isolates were clearly observed by agarose gel electrophoresis. Interestingly, isolates belonging to the same VCG had some different sets of single nucleotide polymorphisms. Twenty-six putative nucleotide polymorphisms were identified among the C. sativus VCGs. Therefore, the results suggested that this marker type may be informative for confirmation the conventionally determined VCGs by increasing the reliability of heterokaryons group classification.