Sugarcane smut disease caused by fungus Sporisorium scitamineum is a major disease and can cause significant yield losses worldwide. In Egypt, this disease is widespread in the commercial sugarcane variety G.T. 54-9. To study the molecular differentiation of S. scitamineum, Five S. scitamineum isolates were collected from five sugarcane production areas in Luxor Governorate (Luxor Grb, EL-Hbeal, EL-Karaia, EL-Mres and Ahaly EL-Mres) in season 2022 and were assessed based on the start codon-targeted (SCoT) marker technique. Four selected SCoT primers amplified 56 DNA fragments, of which 48 (85.71 %) were polymorphic. The polymorphic information content values ranged from 0.240 to 0.393, indicating that a degree of genetic diversity exists among these Egyptian fungusS. scitamineum isolates.Cluster analysis using UPGMA method was divided the S. scitamineum isolates into two main groups, I and II. Group II included only one isolate (isolate 2). Group I was divided into two sub-groups included Sub-G1 (isolates 1 & 3) and Sub-G2 (isolates 4 & 5). The results indicated that diversity of S. scitamineum was partly associated to its geographical origin. These results are useful in breeding programs to obtain smut-resistant sugarcane genotypes. Our results demonstrated that SCoT markers can be used to evaluate the genetic diversity of S. scitamineum.