Macrophomina phaseolina, the causal agent of charcoal root or collar rot, is an important plant pathogen especially in soybean and cotton. Single primers of simple sequence repeats (SSR) or microsatellite markers have been used for the characterization of genetic variability of different populations of M. phaseolina obtained from soybean and cotton grown in India and the USA. Genetic similarity between isolates was calculated, and cluster analysis was used to generate a dendrogram showing relationships between isolates collected from the two hosts. Forty isolates could be clustered into three major groups corresponding to their hosts and geographical region. The wide distribution of microsatellites in M. phaseolina genome was assessed by agarose gel electrophoresis of the PCR products generated by direct amplification of inter SSR regions DNA. This is the first report of the use of microsatellite markers to characterize the charcoal root rot pathogen. The SSR fingerprints (0.25–3.5 kb) generated using DNA from different populations of M. phaseolina of two hosts indicated that these repeats are interspersed within the genome of this pathogen. The variability found within closely related isolates of M. phaseolina indicated that such microsatellites are useful in population studies and represents a step towards identification of potential isolate diagnostic markers specific to soybean and cotton.