Delphinium newtonianum (Ranunculaceae) is a rare endemic of the Interior Highlands. Using a single-copy nuclear gene, genetic diversity was determined for eight populations. Genetic diversity in D. newtonianum is high (37 haplotypes were identified from 80 individuals) and haplotype diversity was 0.885 0.016. Analysis of genetic structure indicates that populations within the Ozark region do not show significant genetic differentiation due to a high level of gene flow. However, Ouachita populations have undergone genetic drift and the loss of genetic diversity relative to Ozark populations. In addition, all Ouachita populations are significantly differentiated from all other populations. A neighbor-joining tree based on p-distance identifies Ouachita populations as a monophyletic group most likely established from a single Ozark population. Ozark and Ouachita populations should be treated as two genetically distinct metapopulations and considered to be evolutionary significant units.