American ginseng (Panax quinquefolius L.), an economically important plant, has been collected and cultivated for use as herbal medicine since the 18th century. A class of triterpenoid saponins, ginsenosides, comprise the most abundant and potent medicinal compounds found in ginseng. This study examined this diversity in the context of the glacial refugia of the Southern Appalachian Mountains for the first time. We hypothesized that 1) chemotypic diversity would be distinct from previously reported values, 2) that plants with a Rg1 dominant profile would be the most commonly observed, and 3) the chemical diversity would be associated with the genetic structure of western North Carolina populations. Seventeen wild ginseng populations in western North Carolina and two from Virginia’s coastal plain were sampled, then six ginsenosides (Rb1, Rb2, Rg1, Re, Rd, and Rc) in the root tissue were extracted and quantified by high-performance liquid chromatography (HPLC), with detection by UV spectroscopy. DNA was extracted from leaf samples and fragment lengths at 7 microsatellite loci were used to analyze genetic diversity. Analysis of these data found Rg1 dominant chemotypes to be the most common in the area studied. These data will assist further examinations of the etiology of chemical diversity of P. quinquefolius, as well as efforts to breed cultivars labeled for their regional origin, phytochemical profiles and specific medicinal properties, ideally reducing the demand for wild harvested ginseng. These analyses lay the groundwork for a more refined characterization of ginsenoside profiles found in wild populations, and support efforts to identify, cultivate, and conserve regional diversity.