Background: Nerium oleander L. plant comprises a large number of components that are beneficial to human health The DNA polymorphism is a useful molecular genetics marker assessment to describe and recognize new germplasm for employing in the crop breeding programs. This study aims at determining the best time durations used to expose Nerium oleander cell suspensions cultures to UV-A rays, for studying differences in the DNA to create new cell lines. Material and Methods: In this study, ISSR was used for the identification of markers associated with the seven treatments exposed to UV-A rays of N. oleander cell suspensions cultures using 10 primers. Results: In the ISSR marker assay, all the primers amplified gave bands, but three primers (UBC810, UBC819 and UBC814) generated low polymorphism, two of them (UBC810 and UBC819) had given low polymorphism percentages (43%) and (19%) respectively and the third primer UBC814 had given a medium polymorphism percentage (57%). The highest value of similarity was 0.950, while the lowest similarity indexed was 0.012. The present study revealed that the Inter-simple sequence repeats   (ISSR) would be a useful assay to identify the genetic variations of the Nerium oleander genome under UV rays stress. Conclusion: This study revealed that the use of ultraviolet rays induces changes in the DNA of N. oleander cell suspensions cultures that produce new genotypes for use in cell lines programs.