Root-rot of cotton (Gossypium spp.) is one of the most important diseases in Upper Egypt. Isolation has been done from diseased cotton roots and seeds which were collected from 11 counties in Assiut province, Egypt. Identification procedures of the isolated fungi confirmed that the isolated fungi were for Fusarium solani, F. moniliforme and F. oxysporum and Rhizoctonia solani. Thirty six isolates of Fusarium spp. and 10 isolates of Rhizoctonia solani were tested for their pathogenicity on both Giza 80 and Giza 83 cotton seedlings to verify their virulence on seedlings. The pathogenicity test results have grouped the Fusarium spp. isolates into three groups; highly virulent that caused 91-100% mortalities; moderately virulent that caused 81-90% mortalities and low virulent that caused lower than 81.0% mortalities. Data also shows that, in general Giza 80 cotton cultivar was more susceptible for infection with Fusarium spp. when compared with Giza 83 cotton cultivar. In case of Rhizoctonia solani, data revealed that the infection percentage was significantly affected by isolates while cotton cultivars had no significant influence on infection. Four 10-mer primers (1:6-d, 2:6-d, 4:6-d and 5:6-d) were used in RAPD-PCR to determine the genetic variability between six isolates, one virulent and one less virulent, of F. moniliforme, F. oxysporum and F. solani. Our results showed that the primer 2:6-d clearly separated F. moniliforme, F. oxysporum and F. solani and proved to be quite powerful in distinguishing the three different species and isolates of Fusarium spp.