Black spot disease of pomegranate is a relatively new disease in Israel that is caused by Alternaria alternata. The symptoms include black spots on leaves and fruit. Only the outer part of the fruit is damaged; the edible tissue remains unaffected. In this study, we obtained 50 isolates of A. alternata from infected pomegranate plants that were classified based on pathogenicity tests using detached leaves. Using an arbitrarily primed polymerase chain reaction, we identified one primer (primer CAG) that reacted only with DNA of isolates that induced the most severe disease symptoms. Based on the sequence of the amplified fragment, we generated a specific primer (primer C) that recognizes these highly virulent isolates. Therefore, we suggest that primer C can be utilized as a molecular marker for the detection of A. alternata isolates that cause black spot disease of pomegranate.