Abstract: Background: Tomato belongs to the Solanaceae family alongside other economically important crops such as pepper, eggplant and potato. Fruit rot disease caused by Alternaria solani is the most severe disease of tomato. Materials and Methods: Seven isolates of Alternaria solani were collected from different tomato fields in diverse localities of Al-Behiera Governorate. Pathogenicity tests of the seven isolates of A. solani were performed on fresh tomato fruits of the 1077 cv. The isolates differed in their virulence as evidenced by the diameters of rotted areas in the inoculated tomato fruits. Results: The isolates 7, 5 and 6 were highly virulent, while the isolates 2 were moderately virulent. The polygalacturonase enzyme activity was examined for all isolates and isolate 7 revealed the maximum activity (1.13 U mL–1 min–1), followed by isolates 5 (1.1 U mL–1 min–1), while the activity was low with isolate 1 (0.42 U mL–1 min–1), isolates 2 and 4 (0.6 and 0.7 U mL–1 min–1), respectively. On the other hand, both of isolates 3 and 6 gave a moderate activity (0.89 and 0.95 U mL–1 min–1), respectively. Genetic diversity was studied using RAPD-PCR and ISSR markers. Results exposed that inclusive genetic variations between the isolates were observed and the polymorphic percentage was ranged from 5.8-80%. While, the ISSR analysis grouped the 7 fungal isolates into two main clusters; the first cluster include isolates 1 and 2 but cluster two was divided into two groups; group one contains isolates 4-6 while group two include isolate 7. Conclusion: The polygalacturonase activity and ISSR gave the same results but the RAPD gave completely different results. That means, using functional gene for differentiation between plentiful closed isolates is more preferable than RAPD. Moreover, the ISSR-PCR is more confident than the RAPD-PCR.