Leaf spot disease caused by Alternaria helianthi (Hansf.) is an important fungal disease of sunflower in India and elsewhere. Non-availability of known sources for resistance to Alternaria is a major constraint in sunflower breed ing. Hence, seeds of sunflower genotypes were subjected to gamma-irradiation to develop mutant populations. Mutants were selected from the M2 generation based on percent disease incidence and were passed on to the M3 generation. Genomic DNA from leaf samples was evaluated by PCR amplification using random decamer primers and the amplified DNA fragments from mutants were compared with their respective controls. Single marker and stepwise regression analysis carried out in relation to percent disease index indicate that the alleles OPC5-B, K, J, OPA12-D and OPA15-A are strongly associated with Alternaria helianthi resistance.