Sequence data from the nuclear internal transcribed spacer (ITS) and the plastid trnL-trnF regions were used to assess relationships among populations of N. odorata across its North American range, and to evaluate whether subsp. odorata and subsp. tuberosa form distinct taxonomic units. Nymphaea mexicana was included because of suspected hybridization with N. odorata. The trnL-trnF region provided a single informative site in N. odorata. In contrast, the ITS region was more variable. Phylogenetic analysis of ITS data supports the monophyly of the two species. Within N. odorata, two clades were resolved largely representing subsp. odorata and subsp. tuberosa, although a few individuals appeared outside the respective clades. Polymorphic sites were detected in ITS, indicating possible hybridization between the subspecies. The geographic location of these hybrids suggests a possible hybrid zone. Overall, molecular evidence supports the segregation of subsp. odorata and subsp. tuberosa, with limited gene flow between them.