Aflatoxins, the most frequently studied mycotoxins, are produced by certain Aspergillus species/strains/isolates of fungi. The aflatoxin biosynthetic pathway studies have led to a number of discoveries. Several structural and their enzymes involved in the biosynthesis of aflatoxins have been discovered and purified. Aflatoxin production and contamination of agricultural crops are major causes of economic losses in agriculture. Thus, better methods of characterization/differentiation are required for both aflatoxigenic and non-aflatoxigenic isolates. Polymerase Chain Reaction (PCR)-based, single sequence repeats (SSR) micro satellites analysis has been used successfully in the analysis of DNA relatedness of species of fungi, bacteria, plants and animals. SSR micro satellites analysis present in nuclear and organelles DNA can be used as molecular markers and has wide ranging applications in the field of genetics including kinship and population studies. These Inter simple sequence repeats (ISSR) represent genome region between micro satellite loci. Sequences amplified by ISSR-PCR can be used for delimiting species. Dendograms which evaluate the likeness between different isolates has also been used.