We used the chloroplast markers trnL-F and rbcL to construct a DNA barcode sequence library of Adiantum (Pteridaceae) from Puerto Rico. By conducting BLAST searches, the trnL-F marker was able to successfully determine specimens of six out of 10 species for which data were generated, and failed for four. For the nine species for which rbcL sequences were obtained, the BLAST search method was successful for three, partially successful for three, and a failure for three. Phylogenetic analyses of both markers were able to resolve and distinguish all sampled taxa, with the exception that sequences of A. latifolium and A. ×variopinnatum were recovered as an unresolved polytomy with A. petiolatum nested within it. The molecular data indicate that what has previously been determined as A. pyramidale on Jamaica, Cuba, Hispaniola, and Puerto Rico likely represents at least two distinct species, based on data from Cuba and Puerto Rico. We also report the presence of A. ×variopinnatum on Puerto Rico for the first time.