Ex situ cultivation in botanic gardens could be one possibility to preserve plant species diversity and genetic variation. However, old ex situ populations are often sparsely documented. We were able to retrieve three different ex situ populations and their source in situ populations of the endangered plant species Silene otites after 20–36 years of isolation. Furthermore, three additional wild populations were included in the analysis. Population genetic diversity and differentiation were analysed using AFLP markers. Genetic variation in the ex situ populations was lower than the variation found in the in situ populations. Strong differentiation (FST = 0.21–0.36) between corresponding in situ and ex situ populations was observed. Bayesian clustering approach also showed a distinct genetic separation between in situ and ex situ populations. The high genetic differentiation and loss of genetic diversity during spatial and temporal isolation in the ex situ populations can be attributable to small population sizes and unconscious selection during cultivation. Therefore, adequate sampling prior to ex situ cultivation and large effective population sizes are important to preserve genetic diversity. Near-natural cultivation allowing for generation overlap and interspecific competition without artificial selection is recommended as being best for the maintenance of the genetic constitution. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, ••, ••–••.