The Vanilla genus is a complex taxonomic group characterized by a vegetative reproduction mode combined with intra- and inter-specific hybridizations, and polyploidy events. These factors strongly impact the diversification of the genus and complicate the delimitation of taxa. Among the hundred Vanilla species, Vanilla planifolia Jacks. ex Andrews and Vanilla × tahitensis J. W. Moore are the main cultivated aromatic species. We applied Genotyping-by-Sequencing to explore the genetic diversity of these two cultivated vanilla species, seven closely related species and nineteen interspecific hybrids. The inter- and intra-specific relationships of 133 vanilla accessions were examined based on 2004 filtered SNPs. Our results showed a strong genetic structuring between the nine species studied, with wild species showing much lower heterozygosity levels than cultivated ones. Moreover, using Bayesian clustering analyses, the kinship of several hybrids could be verified. We evidenced in particular that Vanilla sotoarenasii and Vanilla odorata C.Presl may be the parental species of V. x tahitensis . The analysis of 1129 SNPs for 84 V . planifolia accessions showed a clear genetic demarcation between the vegetatively propagated traditional vanilla cultivars compared to the accessions derived from sexual reproduction, and a higher genetic diversity and lower heterozygosity of the latter (Ho = 0.206) compared to the former (Ho = 0.362). Our data are consistent with a single-step domestication for V. planifolia in accordance with the recent history of its cultivation. It also opens avenues to breed new V. planifolia varieties adapted to biotic and abiotic constraints and to reduce mutational load induced by clonal propagation.