Taxonomists have traditionally distinguished two very similar Southern Appalachian endemic herbs, Micranthes careyana and M. caroliniana, by differences in four floral characters: sepal orientation, filament shape, petal coloration, and fruit length. Yet identification in the field and the herbarium has proven difficult, which is problematic for monitoring populations and determining rarity. The goal of this study was to examine these characters to clarify the differences between these species and their distribution, and to look for molecular differences in DNA sequences. Morphological variation was examined in the field and the herbarium, while leaf material was collected in the field for molecular analyses. Two of the four reported floral characters proved to be useful in species identification: sepal orientation and filament shape. Other key characters were not diagnostic to species. Fixed differences in floral characters were correlated with fixed differences in nuclear and chloroplast DNA sequences, supporting their distinction as unique species in accordance with the diagnosability species concept. In molecular phylogenetic analyses, M. caroliniana and M. careyana accessions are reciprocally monophyletic and may not be sister species. Both are shown to be closely related to M. virginiensis, a widespread and variable taxon. We present a key to identifying M. careyana, M. caroliniana, and M. virginiensis in the Southern Appalachians and lectotypify M. careyana and M. caroliniana, names based on Asa Gray basionyms.