The North American Yellow Lady's Slipper Orchids (Cypripedium) and their relatives present a large amount of variation that has remained intractable to study of morphological characters alone. In this study, the group was investigated using inter-simple sequence repeat (ISSR) markers and morphometric analysis of floral structures. Samples were collected from multiple populations of C. parviflorum and C. pubescens from across their ranges, as well as from populations of C. kentuckiense, C. candidum and C. montanum. One population of C. californicum was included as an outgroup. Individuals were scored for the presence or absence of bands for 8 ISSR primers, and the data were analyzed cladistically using parsimony, and phenetically using UPGMA and neighbor-joining. Twenty-six floral characters were measured and analyzed using principal component analysis; lip shape was quantified using a truss approach. Populations of C. pubescens and C. parviflorum are intermixed in all of the analyses with very few patterns correlated with morphological differences. Some patterns seem to relate to geographical distributions. This could mean either that these morphologies have arisen independently multiple times, or that they were perhaps largely distinct in the past, but continue to exchange genes when in proximity. Populations of C. kentuckiense fall out in two clades associated with two different populations of C. pubescens. This could also be due to convergent evolution or occurrences of secondary hybridization. In each of these cases we favor the secondary gene flow hypothesis. The distinctness of the white lady’s slippers, C. candidum and C. montanum, from the rest of this clade is well supported.

Key words: Cypripedium, ISSR, morphometrics, Orchidaceae