Abstract: Background and Objective: Molecular analysis of sources of resistance to plant pathogens should expedite and confirm novel gene discovery and consequently the development of disease resistant cultivars. By using parents that produce segregating populations with a large genetic variance, breeders could predict which biparental populations will have the maximum genetic variance using the coefficient of parentage or genetic distance estimates based on molecular markers data. To identify molecular markers selection to help soybean breeders in breeding programs and, to study genetic distance among the tested soybean genotypes. Materials and Methods: DNA markers were used as a molecular characterization of genetic improvement of soybean against cotton leaf warm. Four molecular analysis techniques were used in this study to differentiate among 14 genotypes of soybean. First, isozyme variation using native gel electrophoresis (Native-PAGE); secondly, Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) protein electrophoresis for leaves and seeds proteins characterization; thirdly, Random Amplified Polymorphic DNA (RAPD) technique using 10 mer arbitrary primers and fourthly, Restriction Fragment Length Polymorphism (RFLP) using BamHI, MspI and TaqI enzymes. Results: No clear trend has been observed in isozymes analysis regarding differentiation among 14 soybean genotypes based on their resistance to cotton leaf worm. SDS-PAGE for the water-soluble seed proteins in the soybean genotypes exposed a total number of 13 bands most of them are common for the 14 genotypes except two bands with molecular weight 220.36 and 194 KDa for that are specific for Calland genotype (moderately susceptible). Restriction site analysis of the ITS region amplified by PCR using universal primers pair was able to successfully amplify the Internal Transcribed Spacer (ITS) region of all genotypes tested. The RAPD analysis showed that primer A9B7 and A1A13 have 100% polymorphic, however primer A7 and A1 have 0 and 58% polymorphic, respectively. Conclusion: The tolerance was correlated with band density as well as with genotypes, where Forrest, Giza 111, Giza 35, Calland, Clark and Giza 82 were the most related genotypes for tolerant/moderately tolerant against cotton leaf warm. The obtained results could be used as molecular marker selection to help soybean breeders in breeding programs and also, to study genetic distance among the tested soybean genotypes.