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An electrophoretic analysis of allozyme variation from 14 loci in Cypripedium calceolus, C. candidum, C. arietinum, C. acaule, and C. reginae was conducted to address evolutionary conclusions reached in prior studies on the Orchidaceae. The following specific questions were examined: 1) Do the allozyme data suggest that C. arietinum be transferred to the genus Criosanthes? 2) Do Cypripedium populations contain unusually low levels of genetic variation relative to other angiosperms with comparable life histories? and 3) Is the distribution of genetic variation among populations consistent with the idea that evolutionary processes produce higher levels of among‐population differentiation in the Orchidaceae? Very low Nei's genetic identities (0.000–0.285) were found for most species comparisons; however, C. arietinum was not the most genetically distinct taxon and should be retained in the genus Cypripedium. Although most Cypripedium populations contain very low levels of expected heterozygosity, C. calceolus contains unusually high levels in all populations examined. Most species examined here have below average levels of variation distributed among populations. This result indicates that evolutionary processes produce relatively lower, not higher, levels of genetic differentiation among Cypripedium populations compared to other flowering plants. Historical events that could have influenced the observed genetic patterns are discussed.

Cypripedium kentuckiense is a recently described rare orchid found in Arkansas (predominantly) and in eight other states. Much debate has focused on whether this taxon should be recognized as a distinct species or considered to be an extreme manifestation of the variability present in the widespread taxon Cypripedium parviflorum var. pubescens. In this study, 12 isozyme loci were analyzed for 14 populations of C. parviflorum var. pubescens and eight populations of C. kentuckiense. These data were used to examine the genetic similarity of these taxa, assess whether isozyme data support the continued recognition of C. kentuckiense as a distinct species, and assess whether a newly discovered disjunct Virginia population of C. kentuckiense is genetically isolated from other C. kentuckiense populations. The isozyme data revealed that the two taxa are very closely related with a high interspecific genetic identity. However, C. kentuckiense populations contain a subset of the variation present in C. parviflorum var. pubescens, and they have expected levels of heterozygosity that are one-quarter that of C. parviflorum var. pubescens populations. Cypripedium kentuckiense also possesses one widespread unique allele and a unique multilocus genotype. These data suggest that C. kentuckiense should be recognized as a distinct species, possibly of recent origin from C. parviflorum. Lastly, the isozyme data support the hypothesis that gene flow between the Virginia population and other populations of C. kentuckiense has been restricted.