The pantropical genus Vanilla is a member of the Orchidaceae family, one of the largest and most diverse families of angiosperms in the world. It originated in America and differentiated in America, Africa, and Asia. About 132 species of Vanilla have now been accepted since the discovery of the genus. Within the Vanilla genus, leaflessness has appeared at least three independent times, in Africa, Asia, and America as an adaptation to drought conditions. The South-West Indian Ocean (SWIO) region, a biodiversity hotspot for orchids, is home to seven leafless species. Chloroplast markers failed to differentiate species within this recent SWIO clade. Morphological traits used for description are very similar between species and do not provide reliable identification. Moreover, some of the species have overlapping flowering periods and geographic distribution in Madagascar, increasing the probability of finding some sympatric species and, therefore, hybrids. Leafless Vanilla species from the SWIO islands are thus an excellent model for understanding the evolution of orchids. For their conservation, and to confirm the identity of these leafless species, an integrated approach with classical taxonomy using a large number of samples, intense fieldwork on biology and ecology, and molecular studies using variable markers is necessary.

With over 25,000 species, orchids are among families with remarkable high rate of diversification. Since Darwin's time, major advances attributed the exceptional diversity of orchids to plant–pollinator interactions. However, unraveling the processes and factors that determine the phenotypic and genotypic variation of natural orchid populations remains a challenge. Here, we assessed genetic population structure and floral differentiation in recently diverged leafless Vanilla species in a world biodiversity hotspot, Madagascar, using seven microsatellite loci and 26 morphometric variables. Additionally, analyses were performed to test for the occurrence of any patterns of isolation by distance, isolation by environment, and isolation by adaptation and to detect possible physical barriers that might have caused genetic discontinuities between populations. Positive inbreeding coefficients detected in 22 populations were probably due to the presence of null alleles, geitonogamy and/or some admixture (sympatric species). In contrast, the only high‐altitude population showed an important rate of clonality leading to heterozygote excess. Genetic diversity was maximum in western populations, suggesting a postglacial colonization to the north and south. Clustering analyses identified seven genetic groups characterized by specific floral traits that matched five botanical descriptions in the literature. A contribution of montane refugia and river barriers on population differentiation was detected. We also detected combined effects of IBD/IBE and IBE/IBA on genetic differentiation and suggested this pattern is more likely determined by ecological isolation, although pollinator‐mediated divergent selection could not be ruled out for some of the species. Overall, this study provides further insights on speciation in orchids, a group for which Madagascar shows one of the world's highest level of endemism and confirms the importance of the peculiar biogeography of the island in shaping species differentiation.