Seven potentially variable regions of DNA were identified in the plastid genome of Cypripedium calceolus. Five of these were microsatellite-type repeats, one each in the trnL intron, the trnL-trnF intergenic spacer (IGS), the psa1-accD IGS, and two in the rps16 intron. The last two were indels resulting from short tandem repeats in the psa1-accD IGS. Primers were designed for five of these regions, and four produced fragments of variable length. The fifth was invariant. One indel and one microsatellite (both in the psa1-accD IGS) were located in regions with such high A/T content that primers could not be designed. Collectively the four variable DNA regions allowed the identification of 11 different cytotypes in Eurasia, and the data provide strong evidence for the non-native origin of two of the remaining plants in England (both thought to have been planted), and seedlings from these plants are being excluded from reintroduction activities as a result. All five loci have been successfully amplified from DNA extracted from small fragments of tissue taken from herbarium sheets, some more than 120 years old, allowing us to investigate historical patterns. At least three of the loci can be amplified from some other species of Cypripedium.

Key words: biogeography, conservation, Cypripedium, herbarium DNA, plastid microsatellites