Abstract: Background and Objective: Khaya senegalensis (K. senegalensis) A. Juss. (Meliaceae), Known as African mahogany, is a multipurpose timber tree with a variety of economic and environmental values. The aim of the current study was to establish a suitable protocol for K. senegalensis micropropagation. Material and Methods: The effect of benzyl adenine (BA) (0.2, 0.4. 0.6 0.8 and 1.00 mg L–1), types of media [Murashige and Skoog (MS), woody plant medium (WPM) and Gamborg medium] and number of subculture on shootlet proliferation were studied. For rooting stage, indole-3-butaric acid (IBA) (1.0, 2.0 and 4.0 mg L–1) and half salt strengths of both MS and WPM, pre-incubation and incubation time were studied. The PCR, RAPD and ISSR, was used to confirm the true to tip between micropropageted plants and the mother plant. One-way and two-way ANOVA is used for statistical analysis. Results: The results showed the highest number of shootlets per explant (5.33) and the highest number of nodes (22.0) was obtained with 0.6 mg L–1. The BA using half strength of MS culture medium for three subcultures caused the highest number of both shootlets formed per explant and nodes, whereas full strength of WPM culture medium caused the longest shoots and repeated subcultures had inhibit effect on shootlet elongation. For rooting stage, the highest number of roots was obtained with pretreatment of the in vitro shootlets with IBA at 4.0 mg L–1 for one week in darkness, then transplanting them to half MS or WPM for 12 weeks, while the longest roots were obtained with half MS culture medium. Plantlets were successful adapted in peatmoss and sand (1:1). The results of molecular analysis of RAPD and ISSR were obviously revealed that there was no variation between the in vitro derived plants and the mother plant. Conclusion: The current study showed the highest shoot proliferation was recorded with 0.6 mg L–1 and with half MS medium. For rooting stage half MS gave the highest root numbers and length. Molecular marker RAPD and ISSR confirmed that this protocol is suitable for propagation of K. senegalensis with confirmation of true to tip.