The aim of this research was to study the possibility of creating genetic differences in Gardenia plant to obtain a new variety with high economic value. To achieve this goal, three laboratory experiments were conducted as follows: 1-In vitro propagation: The results indicated that the axillary buds when cultured on medium consisting of MS + 0, 5 of BA+ 0.1 mg l -1 of Kin. gave the highest significant results for leaves number 25.4, shoot number 3.28 and shoot length 3.1 cm. The highest significant values of root number and length 6.65 and 1.3 cm, respectively were recorded when shoots were transferred to MS medium combined with 0.75 mg l -1 of IBA. 2-In vitro mutation: Results indicated that the degree of variegated leaves increased by increasing the concentration of the mutagen Ethylmethanesulphonate (EMS) up to 0.4 %, but treatment with a concentration of 0.5% led to explants death or deformation of the leaves. 3-Molecular markers Experiment: An experiment was conducted to examine the applicability of SSR markers to discover the polymorphisms of mutations caused by EMS and their genetic relationships for the first time. Thirteen SSR primers were used. Plants were divided into five groups, namely the original plant and the mutant plants resulting from growth on the five EMS concentrations (0.0, 0.1, 0.2, 0.3 and 0.4%). Cluster analysis was done for all the genotypes under study. Results indicated that 24 bands were obtained, their size ranged between 50: 250 pb, and all of them were 100% Polymorphic. PIC values were calculated, which ranged between 0.32: 0.48 for each primer. H0 was also calculated and the values ranged between 0.32: 0.50. Results in the present study showed the effectiveness of EMS to induce in vitro mutation of Gardenia and this way can be used to develop breeding programs for ornamental plants. However, the use of more than one molecular marker in this type of studies may be more useful to cover multiple regions of the genome.