There is a serious lack of information on the genetic diversity and population dynamics of the 110 tropical Vanilla species, although these are keys elements to adjust conservation strategies. Nineteen polymorphic microsatellite markers were developed from two African leafless Vanilla species V. roscheri and V. humblotii to use in population genetic studies. A transferability analysis of these markers on seven Vanilla species from various geographical origins was conducted. Nine microsatellites were polymorphic in a population of 22 individuals of V. humblotii from Mayotte (Comoros Archipelago). These markers had two to eight alleles per locus and observed heterozygosity ranged from 0.23 to 0.64. Amplification parameters were calibrated to facilitate multiplexing and rapid multi-loci genotyping.