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In Central Europe, Vinca minor has been planted for centuries as an ornamental, medicinal and ritual plant. We asked how variability in genetic and leaf traits of V. minor at ancient localities differs from that at recently established localities sampled mainly in the Czech Republic. Using selected ISSR primers, we obtained two clusters that correspond well with locality history. In the recent cluster, we identified a certain degree of genetic variability, whereas the ancient cluster exhibited none. We recorded significant differences in leaf shape between the clusters. Genotypes with narrow leaves were more characteristic of recent localities. Although the ancient cluster showed no variability in ISSR primers, it did show variability in leaf traits, indicating that some of the morphologically expressed genetic variability cannot be detected using ISSR primers. All samples were diploids (2n = 46), with an identical relative DNA content. Samples from ancient localities, such as deserted medieval settlements and castles, probably descended from a single or a few related clones. This supports the hypothesis concerning the allochthonous character of V. minor in the Czech Republic. (C) 2015 Elsevier Ltd. All rights reserved.