Genomics-based diversity analysis of natural populations is an indispensable tool for enabling informed, advanced, lineage-specific conservation strategies for threatened and endangered species, such as Vanilla barbellata (Orchidaceae). This leafless orchid native to south Florida faces severe environmental threats including rising sea-levels as a result of climate change. A total of 115 individuals from two Floridian populations and three Caribbean populations were sampled. The V. planifolia draft genome was used to identify a filtered set of 12,825 single nucleotide polymorphism (SNP) markers generated via Genotyping-By-Sequencing (GBS). Principal Component Analysis (PCoA) and fastStructure provided resolution of GBS markers confirming distinct populations. Discriminant Principal Components (DAPC), a Neighbor-Joining tree, Diversity statistics and the Fixation Index (Fst) were used to evaluate relationships among and within populations. Floridian populations within the Everglades National Park and the Florida Keys are significantly different and exhibit concerningly low levels of heterozygosity. In contrast, populations of the Guánica State and Susúa State Forests of Puerto Rico have relatively high heterozygosity, but exhibit no discernible gene flow. Finally, the U.S. Virgin Islands population is characterized by an even higher level of heterozygosity, but no gene flow is evident. These results suggest that ex situ conservation of this species is needed because of increased isolation and environmental change and will be best served by targeted collection that captures the greatest level of existing diversity possible.