Determining population genetic structure of isolated or fragmented species is of critical importance when planning a conservation strategy. Knowledge of the genetic composition and differentiation among populations of a rare or threatened species can aid conservation managers in understanding how, and which, populations to protect. The whorled sunflower, Helianthus verticillatus (Asteraceae), is a federally endangered sunflower species endemic to the southeastern United States. The distribution of the species comprises four known populations within three states: Alabama, Tennessee, and Georgia. Recently, new populations were discovered in Marshall County, Mississippi, and Franklin County, Virginia. Here, we carry out a population genetic study of these new populations using nuclear microsatellite markers and compare our results to those from previously known populations of H. verticillatus. Our results show that both newly discovered populations contain novel genetic variation, with Mississippi containing the most private alleles out of all populations tested. The Virginia population is genetically similar to the previously known populations but is under the most conservation concern given the recovery of only two unique genetic individuals found in this population. These results indicate these new populations are worthy of protection and conservation efforts given the unique genetic variation they harbor.