<p><i>Cryptocoryne crispatula</i> var. yunnanensis, an endangered aquatic plant in the Mekong River Basin, is threatened by habitat degradation and population decline. This study aimed to optimize a micropropagation protocol to support ex-situ conservation by evaluating surface sterilization, shoot proliferation, and ex vitro rooting techniques. The most effective sterilization treatment, with 0.01% HgCl₂ for 90&#xa0;min followed by 5% NaOCl for 20&#xa0;min, achieved a 45.45% contamination-free rate. Shoot induction was optimized using Murashige and Skoog (MS) medium supplemented with 0.5 mg L⁻¹ of naphthaleneacetic acid (NAA) and 3.0 mg L⁻¹ of thidiazuron (TDZ), producing 7.14 ± 0.33 shoots per explant. Reducing TDZ to 1.0 mg L⁻¹ during the proliferation phase significantly increased shoot production to 23.75 ± 1.02 shoots per explant. For rooting, in vitro-raised shoots were soaked in a 1X vitamin-enriched MS solution (1X VS-MS) containing 0.5 mg L⁻¹ of indole-3-butyric acid (IBA) for 2&#xa0;h and then transferred to a coarse sand–vermiculite (1:1) substrate. This resulted in 6.31 ± 0.28 roots per shoot and a 73.33% survival rate. Genetic fidelity of the regenerants was confirmed using ISSR markers, indicating clonal uniformity. This streamlined protocol provides a robust platform for ongoing ex-situ conservation and potential habitat restoration efforts for <i>C. crispatula</i> var. yunnanensis.</p> Graphical abstract <p></p>