Thirty five isolates of M. phaseolina, causal organism of Charcoal rot of sorghum, were obtained from different agro-climatic regions of major rabi sorghum growing areas (Karnataka, Maharashtra and AndhraPradesh). Variation among different isolates was studied using PCR-RAPD markers. From a set of 20 random primers tested, a total of 142 amplicon levels were available for analysis. Primers, OPA-03 and OPA-08 showed cent per cent polymorphism. It was possible to discriminate all the isolates with any of the primers employed. UPGMA clustering of data indicated that the isolates showed varied levels of genetic similarity within a range of 0.58 to 0.97 similarity coefficient index indicating significant levels of variation. The study suggested that the grouping of isolates was not related to the regions of collection in anyway. Maximum genetic similarity of 97 % was found between Solapur and Mulegaon. Least genetic similarity was observed between Ahmednagar and Mehboobnagar isolates.