Lionfish (Scorpaenidae) are upper trophic level predators that are native to the Indo-Pacific region and have recently become invasive to the western Atlantic Ocean. Since the invasion was first documented with three individuals in 2000, the number of lionfish in the Atlantic has increased significantly and spurred a series of investigations regarding their biology and potential impacts on the ecosystem. The present study (1) investigates the cause of disagreement between identifications based on meristic and mitochondrial DNA data that were detected for seven native lionfish specimens, (2) determines the number of lionfish species involved in the Atlantic invasion based on genetic data, (3) estimates the extent of the genetic bottleneck that accompanied the invasion and (4) determines the likely source population of the invasive lionfish using mitochondrial DNA haplotypes. The lack of species-specific variation found in fifteen nuclear DNA loci that were examined and the absence of species-specific banding phenotypes resulting from randomly amplified polymorphic DNA (RAPD) analysis leaves the conflict of the meristic and mitochondrial DNA data unresolved. The mitochondrial cytochrome b data, however, indicate that P. volitans along with a small number of P. miles are present in the Atlantic Ocean. The genetic bottleneck that accompanied the invasion resulted in the Atlantic population of Pterois volitans displaying only 10.7% of the genetic diversity found in the native range. The cytochrome b data were also used to identify Indonesia as the most likely area from where the founders of the Atlantic population of P. volitans came.