Evidence shows that dam construction has affected faunal species distribution and diversity in the southeastern U.S. However, the effects of this perturbation on aquatic flora of the region are heretofore uninvestigated. The objective for this research is to examine the effects of hydroelectric power generation and habitat characteristics on the size and distribution of populations of Hymenocallis coronaria (J. LeConte) Kunth (Amaryllidaceae), an emergent aquatic plant found in the southeastern United States, and to characterize its genetic variability in an effort to target conservation priorities. Multiple linear and logistic regression analyses show that hydropeaking does not have a significant effect on H. coronaria populations located downstream of dams, while other habitat variables associated with the streams are significantly positively related to population size and distribution. Analysis of 24 populations using seven universal cpDNA markers and six nDNA microsatellite markers indicates that the genetic structure for both genomes correlates with the location of a major gap in the species’ range, showing deep divergences in haplotype and population dendrograms between eastern and western regions. An AMOVA confirms that a substantial amount of genetic variation is found among regions, and very little variation is found within regions. The eastern and western regions have diverged for a significant time period, potentially due to the presence of a gene flow barrier in the middle of the species’ range caused by sub-standard habitat. Individual drainage basins contain unique cpDNA haplotypes and should be managed appropriately.