Cotton damping-off caused by Rhizoctonia solani is one of the most important and epidemic cotton diseases in cotton-growing regions worldwide, including Egypt. Isolates of Rhizoctonia were collected from infected cotton seedlings showing damping-off symptoms from the different cotton-growing area in Egypt. Twelve isolates of Rhizoctonia were obtained. Pathogenicity test of Twelve isolates of R. solani was evaluated on 5 cotton cultivars, Giza 80, Giza 87, Giza 90, Giza 92 and Giza 93, under greenhouse conditions. All tested isolates were able to infect cotton plants causing damping-off with different percentages of severities, isolate No. R8 and R9 showed significantly highest damping-off percentage, while the lowest percentage of damping-off percentage obtained from isolates No. R11 and R7. For identification of R. solani specific primers were used (ITS1 and GMRS-3) that confirmed that all the isolates were R. solani. The genetic diversity of studied isolates of the pathogen was determined by using random amplified polymorphic DNA (RAPD) markers. A total of 1583 fragments were detected when 18 RAPD markers were applied to 12 R. solani isolates. The phylogenic tree generated from neighboring joint analysis grouped R. solani isolates into groups. The results presented showed that the variability among the isolates of R. solani was high. Variation was found between the polymorphism showed by the isolates which caused the difference in pathogenicity of the isolates.