Nuphar sagittifolia (Nymphaeaceae), Cape Fear spatterdock, is an aquatic macrophyte endemic to the Atlantic Coastal Plain and of conservation concern in North Carolina, South Carolina, and Virginia in the United States. The existence of populations of unclear taxonomic identity has precluded assessment of the number of populations, distribution, and conservation needs of N. sagittifolia. The circumscription of the species was re-assessed using morphological and genetic analyses. Approximately 30 individuals from each of 21 populations of Nuphar across the N. sagittifolia range were included in genetic and morphological analyses, along with the type populations for three taxa. Genetic diversity was assessed by genotyping individuals across 26 SNP loci identified for this study, and morphological variation was assessed through measurement of 31 leaf, flower, and fruit characters. STRUCTURE analysis identified three genetic groups with corresponding morphological differences in the N. sagittifolia range: N. sagittifolia, N. advena subsp. advena, and a third group in the Chowan-Roanoke River drainage that requires further study. A revised key based on Classification and Regression Tree (CART) and Bayesian analyses identifies N. sagittifolia based on a leaf sinus-to-leaf length ratio <0.22, leaves not emergent, and leaf length-to-width ratio usually greater than 2.4. Genetic analyses within the N. sagittifolia group as indicated by the revised key indicated relatively low clonality, low gene flow, and low allelic differentiation among populations. Observed heterozygosity was higher than expected heterozygosity in all populations, an observation consistent with a combination of limited clonal reproduction and sexual reproduction resulting in long-lived genets.