Fusarium oxysporum. f. sp. lycopersici (FOT) causes tomato fusarium wilt, one of the critical fungal diseases causing severe losses in tomato production in Egypt. Eight isolates of F. oxysporum. f. sp. lycopersici were isolated from infected tomato plants collected from six Egyptian governorates. FOT4, FOT2, and FOT5 isolates were highly virulent, while FOT6 and FOT8 were moderately virulent, and FOT7, FOT3, and FOT1 were weakly virulent. All tested ISSRs primers were polymorphic and produced 81 DNA loci of various molecular sizes. DNA fragment patterns analysis showed 37 monomorphic and 44 polymorphic bands, with 54.32% polymorphism between the tested isolates. Among the tested isolates, primer UMC-840 had the highest level of polymorphism (85.71%), while primer HB-13 had the lowest level (33.33%). Dendrogram and ISSR band patterns were analyzed using the UPGMA method to detect the relationship between the eight isolates. The highest genetic homology (82.9%) was observed between FOT4 and FOT3 isolates, while the lowest genetic homology (63.5%) was observed between FOT1 and FOT2 isolates. The eight isolates were classified into three main clusters (A, B, and C). Cluster A includes two sub-clusters; sub-cluster A1 is divided into two groups, group1 consists of two subgroups, subgroup1 include FOT 6 isolate, and subgroup 2 includes FOT4, FOT3, and FOT5 isolates (FOT4 and FOT5 were high virulent isolates), group2 has FOT8 isolate. Sub-cluster A2 includes FOT6 isolate. However, cluster B includes the FOT1 isolate, and cluster C includes the FOT2 isolate.