The red lionfish (Pterois volitans) and devil fire-fish (Pterois miles) are invasive species that pose a threat to the biodiversity and stability of coral reefs in the Western Atlantic, Gulf of Mexico and Caribbean Sea. Species identification of lionfish is uncertain in some parts of Cuba, and research has mainly been focused on their biology and ecology. The principal aim of this study was to determine highly polymorphic markers (Inter Simple Sequence Repeat, ISSR) that could be used in research on lionfish population genetics in addition to confirming the presence of Pterois species in the Guanahacabibes National Park. The genetic profile or “fingerprint” of individuals collected in Mexico, formally identified as P. volitans, was compared with the genetic profile of specimens from Cuba. There were very few “diagnostic bands” and a high number of "common bands", demonstrating that the same species exists in both countries. Furthermore, Nei's genetic distance and the unrooted tree do not show significant differences between both localities. In light of these results, we can confirm the presence of P. volitans in the Guanahacabibes National Park, Cuba. This study demonstrates the functionality of ISSR as a molecular tool for species identification and their application for genetic population studies of this invasive fish species.The red lionfish (Pterois volitans) and devil fire-fish (P. miles) pose a threat to the biodiversity and stability of coral reefs in the Western Atlantic, Gulf of Mexico and Caribbean Sea. Pterois miles has become successfully established on the USA West coast and the island of Bermuda, while P. volitans is more abundant, and has spread throughout the Caribbean reaching as far as Southern Venezuela. Species identification of lionfish is indeterminate in Cuba, and research has mainly been focused on the biology and ecology of the lionfish. Interest in this study was focused on determining highly polymorphic markers (ISSR) that could be used in research on lionfish population genetics as well as confirming the presence of Pterois species in the Guanahacabibes National Park. The genetic profile or "fingerprint" of individuals collected in Mexico, formally identified as P.volitans, was compared with the genetic profile of specimens from Cuba. There were very few "diagnostic bands" and a high number of common bands, which proves that the same species exists in both countries. Furthermore, Nei's genetic distance and the unrooted tree do not show significant differences between both localities. In light of these results, we can confirm the presence of P. volitans in the Guanahacabibes National Park of Cuba. This study demonstrates the functionality of ISSR as molecular tools for species identification and their application for genetic population studies of this invasive fish.