Pollination by water (hydrophily) is a highly specialized mechanism that occurs rarely among aquatic angiosperms, which mainly retain the reproductive systems reminiscent of their terrestrial ancestors. Hydrophily is abiotic and typically associated with unisexual flowers, factors that predictably would promote xenogamy and outcrossing. Yet, there have been few reports of hybridization involving waterpollinated plants (hydrophiles), with no firm evidence of natural interspecific hybridization. The genus Najas comprises about 40 species of submersed aquatic plants, all characterized by subsurface hydrophily. Hybridization in this genus has been suspected, but verified previously only among infraspecific taxa. In this study we document the first instance of interspecific hybridization in Najas using genetic evidence from three populations that were identifiable as N. guadalupensis but yielded polymorphic DNA sequence profiles. To facilitate our analysis we first conducted a phylogenetic survey of New World Najas taxa using nuclear and chloroplast markers. Alleles cloned from a biparentally-inherited locus (ITS) in these aberrant populations associated with two distinct but phylogenetically sister species (N. guadalupensis subsp. olivacea and N. flexilis) thus confirming their hybrid origin. In all cases the chloroplast markers associated with N. guadalupensis subsp. olivacea, implicating it as the maternal parent. The hybrid Najas plants occur at the edge of the sympatric range of the parental species. They possess no readily distinctive morphological features and require genetic analysis for confident detection. One population grows aggressively, raising concerns that at least some hybrid Najas plants represent a potential conservation threat. The possible hybrid ancestry of the endemic N. guadalupensis subsp. muenscheri also was assessed, but could not be confirmed or refuted by the data evaluated.