Thirty Fusarium oxysporum f. sp. cucumerinum (FOC) isolates were collected from Kafr El-Sheikh governorate, Egypt to study their pathogenic variability and genetic diversity. All isolates caused the typical symptoms of wilting with different severity degrees and reduced cucumber growth parameters and crop productivity under greenhouse conditions. We have classified the isolates to different groups based on pathogenicity, morphological characters, disease quantum degrees and genetic variation. The polymorphisms obtained from RAPD and protein profiles revealed a high degree of genetic diversity and their cluster analysis was matched with the results obtained from the pathogenicity. Previously it was believed that the different collection regions of the same pathogen isolates were the cause for their pathological and morphological differences, and there was no genetic variation can be occurring between isolates as long as they were collected from the same place. In our results, the genetic diversity of FOC isolates proves that the genetic variation of fungus isolates can occur in the same geographical location.