In this investigation, three drainage water samples were collected from selected agricultural wells in Dakhalia and Kafr El–Sheikh Governorates. The drainage water samples were analyzed for physico – chemical properties. Six ISSR primers were used to assesst genetic diversity induced on the molecular level due to the treatments of onion roots with wastewaters from the different resources. This technique exhibits a great diversity in onion genotype particularly on the molecular level. The purpose of the present investigation was to evaluate the level of water quality on the molecular level using ISSR biomarkers, in addition to compare chemical composition of drainage water with the standards of WHO guidelines for drinking water quality. Onion roots , as biological indicator can be used to measure the genotoxic effects of water pollutants on DNA molecule via measuring the polymorphism of DNA fragments in treated roots. Results suggested that a qualitative measurement revealed changes in ISSR profiles. A distinct distance appeared between the band profile of treated roots and control samples. The comparison between treated and untreated genomes revealed that ISSR analysis could be used as a new tool to evaluate the level of water quality through how the drainage water modifies the structure of DNA molecule in living organisms. This study indicated that extreme application of chemical fertilizers had a severe impact on water quality. The physic-chemical analysis of drainage water quality used in this study revealed that the drainage water quality does not meet the WHO standards about direct reusing it in irrigation.