Allozyme and RAPD analyses conducted on Cypripedium reginae populations from previously glaciated and unglaciated sites indicated low species- and population-level diversity and moderate population differentiation. Low species-level variation may be attributed to founder effect during Pleistocene range shifts, genetic bottlenecks in a relatively small refugial southern range, and genetic drift in small and isolated southern populations during glacial advance. Populations from previously glaciated sites harbored higher genetic diversity than populations from unglaciated sites. This geographic pattern of population genetic structure is highly irregular among organisms with disjunct ranges where most studies have revealed higher levels of genetic diversity in unglaciated refugial populations. We attribute this pattern in C. reginae to the presence of abundant open wetland habitat near advancing glaciers that served as refugia for diverse northern populations that were well positioned to recolonize open wetland habitat after final recession of Pleistocene glaciers.