Tomato wilt disease is one of the most destructive fungal diseases infecting tomato plants in Egypt. The disease is caused by Fusarium oxysporum f. sp. lycopersici (FOL), a notorious pathogen that colonises xylem vessels of its host causing wilt besides other disease-associated symptoms. In total, eighteen different isolates of FOL were recovered from specimens collected from characteristic symptom-expressing tomato plants growing in five different governorates of Egypt, namely, Alexandria, El-Beheira, Kafr El-Sheikh, Giza and Assiut. Of these isolates, 13 were obtained from the root, 4 from the rhizosphere and one from the stem. Isolates were identified based on their cultural and microscopic morphological features. The pathogenicity of the eighteen isolates was tested against four different commercial tomato varieties (030, Dussehra, 86, and 111) in Egypt. Two isolates (FOL4013 and FOL4014) from Giza showed high virulence, while three isolates (FOL3010, FOL3011 and FOL3112) from Kafr El-Sheikh exhibited low virulence. The isolates from El-Beheira and Assiut showed intermediate virulence grades across the spectrum. The radial growth rate of the mycelium and conidia sporulation were assessed for all isolates. Radial growth rate ranged from 0.6 to 0.9 cm/24h with conidia sporulation rate ranging from 1.2 to 2 spore per 10 cm³ (x10⁶) of the suspension. The variation in rates among different strains was statistically significant at P≥0.05. Five of the studied isolates were molecularly characterised using PCR amplification of the ITS region followed by DNA sequencing to identify their genetic relationship and the corresponding phylogenetic tree was graphically presented.