The federally listed threatened species, Helenium virginicum, is endemic to 30 sinkhole ponds in two counties in Virginia, but a Helenium population in Pomona, Missouri, differs scarcely from H. virginicum from Virginia in morphology, ecology, and non-coding nrDNA ITS sequences. In response to continuing taxonomic uncertainty regarding the Missouri population, we enlarged our ITS comparisons from our previous work to include 48 populations, more than doubling the number of H. virginicum populations, and including populations of H. autumnale and H. flexuosum from 12 states in the United States and two Canadian provinces. Phylogenetic analyses were conducted using Bayesian, maximum-likelihood, and maximum-parsimony methods, and showed strong support for a monophyletic species of all H. virginicum from Virginia, the Pomona population and 29 other recently discovered populations in Missouri. We conclude that H. virginicum is narrowly endemic and disjunct between Virginia and Missouri. Additionally, one population of H. autumnale from the Bruce Peninsula, Canada, was found to be the sister group to H. virginicum. We discuss the impact of Pleistocene and Holocene geoclimatic changes on the phylogeography of these plants, including the possibility that the H. autumnale complex has become more disjunct and narrowly endemic since the Xerothermic Period (8–4Ka).