Macrophomina phaseolina (Tassi) Goid is one of the most important soil-borne fungi that affects sesame and causes charcoal rot disease, with a great economic challenge to sesame growers worldwide. Pathogenic and molecular characterizations of eleven M. phaseolina isolates collected from different geographical regions in Egypt were carried out to determine the pathogenicity and genetic diversity. Pathogenicity tests showed a pathogenic variability among the isolates. The identification of M. phaseolina isolates was confirmed by a specific primer and analyzed for genetic diversity using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. The correlation between RAPD analysis of M. phaseolina isolates and their pathogenicity were estimated. Cluster analysis placed the isolates in two distinct clusters and exhibited clear correlation with their pathogenicity. Analysis of ISSR profiles revealed distinct genetic diversity among isolates and showed different clusters according to the geographical regions, in which close geographic origins tend to group nearly. The present study clearly demonstrated that M. phaseolina isolates which were obtained from different geographical regions were highly variable. RAPD and ISSR markers were suitable to reflect the genetic diversity among the studied isolates and could help in DNA finger printing which can be used in future breeding programs of sesame.