The molecular study is based on genomic DNA therefore DNA must be extracted in a pure and uncontaminated form. In this study, genomic DNA of twenty four isolates of Trichoderma species (T. harzianum, T. hamatum, T. koningii and T. pseudokoningii) indicated band of ~15Kb on 0.8% agarose gel and quality was determined by obtaining absorbance ratio (260/280) in the range of 1.7-1.9. Restriction fragment length polymorphism (RFLP) analyses were performed by using two restriction endonulease enzymes i.e., BamHI and HindIII. The BamHI represented results in the range of 500bp-750bp. 18S rRNA gene targeting was further carried out through optimization in ribotyping analysis.