This is the first report evaluating the genetic diversity of Mexican grape species utilizing DNA-based markers to understand the distribution of grape species and patterns of hybridization. The study utilized accessions maintained in three collections in Mexico, one in the USA and recently collected germplasm. Fifteen SSR markers were used to develop a fingerprint database to identify unique germplasm. Two different clustering analyses without prior population assignment, were used to identify groups that were verified by a Discriminant Analysis of Principal Components and a Principal Coordinate Analysis. Genetic diversity estimates were made across and within groups to validate the results obtained from the clustering analyses. Multiple clustering analyses and diversity parameters supported six genetic groups representing different geographic regions. The Northeastern group was the most diverse with a geographic range extending to Eastern and Central Mexico, while the Coahuila group was the least diverse. Vitis arizonica Engelm. and V. cinerea Engelm. ex Millardet were the most abundant species with many hybrid forms. We provide evidence that wild grape species in Mexico follow the physical barriers of mountain ranges like the Sierra Madre Oriental with an east-west divide and the Trans-Mexican Volcanic Belt as a corridor for gene flow among different grape species. Additional collections are required to fully understand the extent of hybridization and to clarify hybrid zones.