Abstract Helenium virginicum is a narrowly endemic federally protected species, disjunct between Virginia and Missouri. To study the magnitude and distribution of genetic diversity within this species, we determined ISSR fingerprints and cpDNA haplotypes of plants in nine Virginia and eight Missouri populations. We found high ISSR diversity at the species level, with higher diversity in Virginia than Missouri. Additionally, moderately low diversity at the population level and high population structure suggests low gene flow among populations. Mean divergence between Virginia and Missouri populations was greater than between populations in either region. Missouri populations were more structured than Virginia populations. We found six distinct cpDNA haplotypes distributed among H. virginicum populations, with one found in both regions, two only in Virginia, and three only in Missouri. High genetic divergence among populations and between regions, the demographic asynchrony of populations, and self-incompat...