Morphology, species delimitation, and interspecific relationships were evaluated in a phylogenetic context in the aquatic monocotyledon genus Vallisneria using a combination of morphological and molecular (nrITS, rbcL, trnK5' intron) data. Contrary to previous studies that recognized few species worldwide, we distinguished 12 species by molecular data, and an additional 2-3 species by morphological differences within groups that were invariant at the molecular level. Two new Vallisneria species (V. australis, V. erecta) are formally described. Other potentially novel species were detected from the cultivated material examined but require further study to elucidate their taxonomic status. Phylogenetic analyses indicated that vittate (caulescent) species (including Maidenia rubra) are not basal, but nested between two groups of rosulate (rosette) species. To preserve Vallisneria as monophyletic, a new combination is made (V. rubra) that accommodates the transfer of M. rubra to Vallisneria. Several taxonomic characters associated with the stigma morphology of pistillate flowers were found to represent suites of features related to pollination. In most cases, these character suites corresponded to a particular arrangement of filaments in the staminate flowers. The precise geographical origin of Vallisneria remains difficult to determine. However, we conclusively documented the presence of the Old World V. spiralis in Texas (United States), which constitutes the first authentic record of this nonindigenous species in North America.