This study aimed to testing water quality on the molecular level using ISSR markers via genetic variation induced in the genome affected by the testing water sample.  Inter–simple sequence repeats (ISSR markers) are the area in the onion genome flanked by microsatellite sequences. The amplification of these regions via PCR using a single primer produced multiple amplification products which could be used as a dominant multilocus genetic marker for studying the genetic diversity in various treatments. Microsatellites are the regions in the genome that consists of short DNA motifs as usually 2 – 5 nucleotides long which repeated linearly multiple times. In this study three water effluents collected from different water resources in addition to the control water from Nile River were used for testing their genetic effects on the onion genome. Six molecular markers were used in this study to achieve genetic modification induced among the four treatments of water effluents on onion roots. The amplified products varied in size from 150 – 1200 bp. ISSR – 3 primer showed that the poor quality water resulted from fertilizer industry which generated a lower number of bands followed by Menyet  El Nasr and Kafr El – Sheikh drainage waters, respectively. The better quality water obtained from the Nile River which generated the highest number of bands in relation to other water effluents from different resources. This indicated that the total number of generated fragments was associated with better water quality.