Menyanthaceae consist of five genera of aquatic and wetland plants distributed worldwide. The three monotypic genera (Liparophyllum, Menyanthes, and Nephrophyllidium) are clearly differentiated morphologically, but the two larger genera (Nymphoides and Villarsia) contain several taxa of uncertain affinity. We undertook a phylogenetic analysis, using a combination of morphological and molecular data, to resolve relationships among species and to evaluate the current circumscription of genera. DNA sequence data for nuclear (ITS) and chloroplast (rbcL and trnK/matK) gene regions were largely congruent (by partition-homogeneity test), and a combined data phylogeny revealed several strongly supported relationships. Analyses using asterid outgroup taxa supported the monophyly of Menyanthaceae. Menyanthes trifoliata and Nephrophyllidium crista-galli comprised a clade sister to the remainder of the family. Species of Nymphoides, except N. exigua, resolved to a single, deeply-nested clade, indicating that the floating-leaved habit is derived evolutionarily within the family. The genus Villarsia comprised a paraphyletic grade toward Nymphoides, wherein the species resolved to three assemblages: (1) a shallowly nested clade containing V. albiflora, V. calthifolia, V. marchantii, V. parnassifolia, V. reniformis, and V. umbricola; (2) an isolated South African clade including V. manningiana and the type species, V. capensis; and (3) a heterogeneous clade of taxa from three genera, including V. exaltata, V. lasiosperma, and V. latifolia, plus the anomalous species V. capitata, V. congestiflora, Liparophyllum gunnii, and Nymphoides exigua. Our results indicate that the genera Menyanthes, Nephrophyllidium, and Nymphoides should be retained as circumscribed, with the exception that Nymphoides exigua should be restored to Villarsia. The genus Villarsia, however, eventually should be subdivided among monophyletic lineages, whereby in the strict sense Villarsia would contain only South African taxa.