Invasive species are one of the largest threats to vulnerable ecological communities and biodiversity today and are economic burdens across the globe. It is therefore crucial that we understand the origins and the driving forces that promote the establishment, persistence, and spread of these taxa. Polyploidy, or whole genome duplication, has been suggested as a possible factor facilitating the success of invasive taxa, yet is an understudied aspect in invasion biology. Although ferns are often neglected in invasive species inventories, several fern families are overrepresented as naturalized and invasive taxa including the vining ferns in the family Lygodiaceae. The Japanese climbing fern, Lygodium japonicum, is native to eastern Asia, and since its introduction in the early 1900s through the ornamental plant trade, it has rapidly spread throughout the southeastern United States, creating dense thickets that smother native plants and disrupt agricultural pine logging. While previous chromosome counts of L. japonicum suggest that both diploid and tetraploid cytotypes occur in its native range, there are no data for populations in the invaded range to date. Using chromosome counts, flow cytometry, and spore size measurements, we assessed the ploidy of invasive populations of L. japonicum in the state of Florida. We found that L. japonicum is represented by a tetraploid cytotype throughout Florida. Our study is the first to examine the ploidy of invasive L. japonicum populations, although additional work will be needed to determine if this species is tetraploid throughout its invaded range.