The floating-leaved aquatic plant genus Nymphoides includes diploid and polyploid species that are distributed worldwide. Prior studies found widespread incongruence between nuclear and plastid phylogenies, and this has prevented the construction of a unified phylogenetic hypothesis for the genus. In order to examine whether phylogenetic incongruence resulted from ancestral polyploidization events, we obtained DNA sequence data from two nuclear loci, phytoene desaturase (PDS) and transmembrane nine (TMN1), as well as the plastid trnT—trnF region. Multiple divergent copies of the nuclear genes were recovered from several species, and species with the most divergent copies were inferred to have originated through allopolyploidization. Our data support a scenario in which three groups of species descended from allopolyploidization events: a clade of North American species (N. aquatica and N. cordata), a clade of neotropical species (N. fallax, N. grayana, and N. humboldtiana), and the Australian species N. montana. Trees constructed using novel nuclear gene data also were incongruent with previously generated nuclear and plastid data, however we were able to combine data from four independently evolving regions (PDS, TMN1, nuclear internal transcribed spacer, and plastid) to generate a single species tree that received strong support at several nodes. Sources of incongruence on the species tree involved a relatively small subset of species. Overall, the increased data from nuclear loci have dramatically improved our understanding of the Nymphoides phylogeny.