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Olea europaea is an economically important tree species native to Mediterranean region. The present study is focused on developing the regeneration protocol and production of true-to-type plants of Olea europaea L. cv Barnea. Nodes from adventitious shoots were surface sterilized and inoculated in different tissue culture media such as Woody Plant Media (WPM), Murashige and Skoog media (MS), Rugini Olive media (OM), Schenk and Hildebrandt media (SH) and Multiplication media (Combination of half strength MS and half strength Rugini OM) in which culture establishment was best obtained in multiplication media supplemented with 1mg/l zeatin. The explants treated with fungicide in addition to other surface sterilants resulted in reduced in-vitro explant contamination. Different treatment was given to induce shoot proliferation and the highest shoot number 4.2±0.4 was obtained in multiplication media enriched with 2.0 mg/l zeatin and silver nitrate at 2.0 mg/l. The effect of sub-culturing on shoot proliferation was also studied. The in-vitro derived shoots were assessed for genetic fidelity using different molecular markers Random Amplification of Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). Monomorphic bands were obtained between the mother DNA and in-vitro derived progenies which indicated that there is genetic stability in the in-vitro regenerated plants. The protocol developed shows the highest multiplication rate and confirms genetic uniformity by assessing through RAPD and ISSR markers. In this study, for the first time we described a successful in-vitro regeneration protocol for uniform plant production of Olea europaea L. cv. Barnea.