The rhizostome jellyfish Mastigias (Agassiz, 1862), native to the western and central Indo-Pacific, is recorded from the western Atlantic, in a marine lake in Puerto Rico (Laguna Joyuda) and a working quarry on No Name Key, Florida (USA) in the Florida Keys. The single individual from Laguna Joyuda, collected in 2002, was previously misidentified as Phyllorhiza (Agassiz, 1862) and Mastigias has not since been recorded there, while a large population of Mastigias was observed in No Name Key in 2009 and 2010 and may have been present for decades. Identification as Mastigias for both sampling sites was confirmed by molecular systematic analysis of mitochondrial cytochrome c oxidase I (COI) and ribosomal 16S (16S). Sequences from both molecular markers were identical for all individuals sampled from Puerto Rico and the Florida Keys. Phylogenetic analysis of COI indicated that the introduced Mastigias were most closely related to Mastigias sp. 1 (Dawson, 2005) from Kakaban, Indonesia (Kimura 2-parameter sequence divergence = 1.1%) and distantly related to Mastigias papua (Lesson, 1830) from Palau (6.4-7.2%) and Mastigias sp. 2 (Dawson, 2005) from Papua New Guinea (9.8%). Therefore, the source region may lie somewhere in the central Indo-Malayan region, though determining an exact source region is not possible at this time. While the invasion vector is unclear, possible mechanisms include commercial shipping, mobile oil platforms and live rock transport. These are the first two confirmed records of Mastigias in the Atlantic basin and the second confirmed identification of the genus occurring outside of the native range, after Hawaii.