Nymphaea odorata, Nymphaeaceae, is the most widely distributed water-lily in North America. Disagreement exists on whether this morphologically variable species should be split into two species, N. odorata and N. tuberosa, or treated as one species with two subspecies. Morphological characters and markers from the inter-simple sequence repeats (ISSRs) were examined to assess taxonomic status and elucidate patterns of genetic variation among populations. This study provides evidence against treatment of N. tuberosa at species rank. The principal component analysis of 26 vegetative characters underscores immense variability, but does partially segregate populations of subsp. odorata and subsp. tuberosa. Based on analysis of variance, a new set of morphological characters is proposed to distinguish the two subspecies: mean leaf blade length-to-width ratio, petiole striping, and lobe apex shape. Results from ISSRs show high polymorphism within and among populations. Genetic variation was found largely within geographical regions (89%) rather than among regions. Principal coordinate (PCOA) analyses and minimum spanning tree (MST) analyses based on ISSRs clearly distinguished Nymphaea mexicana and N. odorata. Within N. odorata, samples of subsp. odorata appear to be a distinct entity, whereas samples largely but not completely separated from samples of subsp. tuberosa. PCOA and MST showed linkage between most samples of subsp. odorata whereas this was less evident in UPGMA.