Vanilla planifolia Jacks. is a species of great economic importance, since vanillin, a compound highly valued in the food and pharmaceutical industry, is extracted from its pods. This species is in the category of special protection, so it is important to take actions for its conservation and to maintain the genetic stability of the conserved germplasm. An adequate way to achieve this is through the minimal growth in vitro conservation techniques. The present work aimed to establish an in vitro conservation protocol for vanilla germplasm that allows the genetic stability of the conserved material. For the establishment of the minimal growth in vitro conservation protocol: two concentrations of basal Murashige and Skoog (MS) medium (50% and 100%), two incubation temperatures (4 and 22 °C) and two concentrations of abscisic acid (ABA) (3 and 5 mg⋅L ⁻¹ ) were evaluated. To evaluate the genetic stability of the germplasms used in this study (cultivated, wild, and V . insignis morphotypes) by analyzing the profiles of molecular markers SSR (simple sequence repeats) and ISSR (inter simple sequence repeats). The MS medium (100%) supplemented with 3 mg⋅L ⁻¹ of ABA and incubated at 22 °C, was the best treatment for the in vitro conservation of Vanilla spp. Compared with the control treatment, it allowed us to obtain smaller shoots (1.17 × 0.17 cm), which showed high genetic stability, given by the low percentages of polymorphism detected in morphotypes cultivated and wild (SSR 0%, ISSR 2%) and V. insignis (SSR 0%, ISSR 0%). We conclude the usefulness of the established protocol to conserve the genetic variation of the evaluated Vanilla germplasm.