Molecular variability of vanilla (Vanilla planifolia) and three wild species was assessed to explore the possible sources of variation that can be used for crop improvement. A total of 154 ISSR loci were analysed by the UPGMA, assignment tests of individuals (STRUCTURE) and indices of genetic diversity. The assignment tests were done at two levels: first considering the four species and then only the accessions of V. planifolia. The molecular analysis indicated 99.3% polymorphism among all species and 70.45% within V. planifolia. The UPGMA showed the separation of these four species into three groups and grouped V. planifolia accessions into three subgroups. The more genetically differentiated accessions were of the Rayada morphotype and a wild accession was from Oaxaca, followed by a wild accession from Quintana Roo; all the commercial accessions of V. planifolia (Mansa morphotype) were grouped together. The STRUCTURE analysis differentiated between V. planifolia and the three wild species, and among the accessions of the Mansa and Rayada morphotypes and the wild accessions. The STRUCTURE analysis also indicated the presence of mixed individuals. These results are of great importance since the accessions of V. planifolia that are genetically more differentiated are the most threatened due to the scarcity of these individuals, the destruction of habitat and replacement by the commercial morphotype. These individuals should be salvaged and used to expand the genetic background of vanilla.