Demand for all-natural vanilla flavor is increasing, but its botanical source, Vanilla planifolia, faces critical challenges arising from a narrow germplasm base and supply limitations. Genomics tools are the key to overcoming these limitations by enabling advanced genetics and plant breeding for new cultivars with improved yield and quality. The objective of this work was to establish the genomic resources needed to facilitate analysis of diversity among Vanilla accessions and to provide a resource to analyze other Vanilla collections. A V. planifolia draft genome was assembled and used to identify 521,732 single nucleotide polymorphism (SNP) markers using Genotyping-By-Sequencing (GBS). The draft genome had a size of 2.20 Gb representing 97% of the estimated genome size. A filtered set of 5,082 SNPs was used to genotype a living collection of 112 Vanilla accessions from 23 species including native Florida species. Principal component analysis of the genetic distances, population structure, and the maternally inherited rbcL gene identified putative hybrids, misidentified accessions, significant diversity within V. planifolia, and evidence for 12 clusters that separate accessions by species. These results validate the efficiency of genomics-based tools to characterize and identify genetic diversity in Vanilla and provide a significant tool for genomics-assisted plant breeding.