The root and stem rot caused by Macrophomina phaseolina (Tassi.) Goid. is most important disease of sesame and its causing 5-100% yield losses. Fifteen isolates of Macrophomina phaseolina collected from different major sesame growing areas of India. In cultural and morphological variability, the mycelia growth (fluffy, submerged & partially submerged), colony colour (black, grayish white, blackish grey, grayish black), colony texture (cottony & velvety), and colony diameter (75 to 90mm), sclerotia weight (0.05 to 0.32 mg), intensity (11.66 to 84.66/100X magnification), shape (oblong, irregular & round), distribution (scattered & uniform) and size ranged between 97.858 x 70.382 to 201.880 x 140.169 µm. Among the fifteen isolates of M. phaseolina, MP-1 (Jabalpur, Madhya Pradesh) isolate of M. phaseolina was more virulent and aggressive as it produced maximum intensity of sclerotia per microscopic field and MP-13 was least virulent having maximum size (201.880 µm x 140.169 µm). Pathogenic variability among 15 isolates of M. phaseolina through cut stem inoculation method was performed for all the isolates of M. phaseolina and MP-1 (Jabalpur, Madhya Pradesh) isolate causes mean maximum linear stem necrosis (13.96 cm) on different susceptible varieties of sesame, and considered as most virulent and aggressive isolate. Molecular identification and its variability in fifteen isolates of M. phaseolina using universal fungal ITS 1 (forward) & ITS 4 (reverse) primer. Sequences of the ITS region of all the isolates (MP-1 to MP-15) showed 100 percent identity to each other and all the isolates are 95-99 percent similar when compared to a M. phaseolina ITS sequence reported in GenBank (NCBI), which indicates that all the isolates were identified as M. phaseolina. A potential pathogen is often blessed with biodiversity within its population. Basically, variation in pathogen is desirable trait for its existence in nature. This variability among the pathogens underlies their ability to withstand the host environment and its interaction.