Alternaria black spot disease on pecan is caused by the opportunistic pathogenAlternaria alternataand poses a serious threat to the local South African and global pecan industry. Several diagnostic molecular marker applications have been established and used in the screening of various fungal diseases worldwide. The present study investigated the potential for polymorphism within samples ofA. alternataisolates obtained from eight different geographical locations in South Africa. Pecan (Carya illinoinensis) leaves, shoots, and nuts-in-shuck with Alternaria black spot disease were sampled, and 222A. alternataisolates were retrieved. For rapid screening to identify Alternaria black spot pathogens, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis of the Alternaria major allergen (Alt a1) gene region was used, followed by the digestion of the amplicons withHaeIII andHinfI endonucleases. The assay resulted in five (HaeIII) and two (HinfI) band patterns. Unique banding patterns from the two endonucleases showed the best profile and isolates were grouped into six clusters using a UPGMA (unweighted pair group method with arithmetic averages) distance matrix (Euclidean) dendrogram method on R-Studio. The analysis confirmed that the genetic diversity ofA. alternatadoes not depend on host tissues or the pecan cultivation region. The grouping of selected isolates was confirmed by DNA sequence analysis. TheAlt a1phylogeny corroborated no speciation within the dendrogram groups and showed 98–100% bootstrap similarity. This study reports the first documented rapid and reliable technique for routine screening identification of pathogens causing Alternaria black spot in South Africa.