Roots of Zeyheria montana, a species native to the savanna (Cerrado) region of central Brazil, produce lapachol, a naphthoquinone with anticancer activity. Lapachol is also the precursor of β-lapachone, a novel drug candidate for preventive and adjuvant cancer therapies. The leaves of Z. montana are a renewable source of ursolic acid and oleanoic acid, compounds known for their anticancer, antioxidant and antimicrobial properties. The potential prophylactic use of β-lapachone, as well as the medicinal properties of ursolic acid, highlights the importance of this study on Z. montana's germplasm conservation. Multiple shoots were induced on Woody Plant media with supplemented 0.1 mg·L −1 of thidiazuron (TDZ). Rooting was promoted on half strength WP (Woody Plant) media containing 1.0 mg·L −1 of indole-3-butyric acid (IBA). Plantlet acclimatization to ex-vitro condition was done at a 70% success rate using different substrates. It was possible to store Z. montana's elite germplasm using in vitro cultures of media containing 2% sucrose plus 4% sorbitol for six months without subcultures.