Root rot is a serious threat to the sesame crop, causing a significant yield loss in Odisha, India. Ten isolates were recovered from different geographical locations in Odisha, India, to understand the relationship among phenotypic characteristics, virulence, and genetic base of the pathogen Macrophomina phaseolina. Genetic diversity analysis using four ISSR primers generated twenty-seven bands with 71.9% polymorphism. The PIC value ranged from 0.59 to 0.663, with a maximum PIC value in ISSR9. UPGMA grouped isolates into three distinct clusters with 60 % genetic similarity. The ordination of isolates in the dendrogram and PCA analysis showed a consistent relationship of geographic origin with the genetic base of the pathogen. A similar grouping pattern was observed based on phenotypic traits and virulence of isolates. All isolates exhibited a significant variation in phenotype (colony colour, colony type, growth rate, aerial mycelium microsclerotia size, shape, colour, and abundance) and virulence (against cultivar VRI- 1). In addition, a negative correlation was observed between the sizes of microsclerotia with virulence. The findings confirm a considerable variation among the isolates and a strong relationship between phenotypic characteristics, virulence, and the genetic base of the pathogen. Knowledge of these characteristics may help to understand the population structure of the pathogen.