Sorghum is the second African most important cereal, it's well adapted to tropical and sub-tropical areas, It enters in the industry of flour, fuel, source of fiber, the manufacture of fermentation and beer, extracting some chemical compounds like dextrin, windbreakers, building fences and fresh fodder crops especially in summer. Microsatellites or Inter Simple Sequence Repeats (ISSRs) are becoming the markers of choice in many plant breeding programs because they are transferable, multiallelic co-dominant, PCR-based, easily reproducible, randomly and widely distributed along the genome. Twenty Egyptian sorghum (Sorghum bicolor (L.) Monech) genotypes were assessed for DNA polymorphism using 10 ISSR primers. All tested ISSR primers yielded amplified products and generated 152 alleles (average of 15.2 alleles/marker). Out of the 10 used ISSR primers, eight primers obtained 24 unique markers among the 20 studied sorghum genotypes. However, molecular characterization revealed polymorphism percentage of a 100% for ISSR markers, including a high level of polymorphism among the studied sorghum genotypes. On the other hand, cluster analysis using UPGMA method classified the 20 sorghum genotypes into two groups using ISSR markers. The cluster analysis showed a high genetic variation among the studied sorghum genotypes and the diversity of these genotypes was in an agreement with their source pedigree. The results of principle coordinate analysis (PCoA) were closely in line with those of the cluster analysis. These results could be used by breeders to develop protocols for sorghum improvement. From the commercial point of view, molecular fingerprinting is a useful tool for several applications, including its use in breeding programs, in determining relatedness of genotypes and for pedigree verification. Molecular fingerprints are useful for varietal protection to prove ownership or derivation of plant lines as well as to prove violations and breaching of patent and plant variety protection certificates or the misappropriation of trade secrets.