Many botanic gardens keep ex situ collections of rare species to prevent their extinction and to enable their reintroduction into the wild. A potential problem with ex situ collections is that relaxed selection, genetic drift, novel selection and inbreeding may cause rapid loss of adaptation to natural conditions and therefore may hamper success of reintroductions. Here, we investigated whether cultivation in ex situ collections of three threatened species— Trifolium spadiceum , Sisymbrium austriacum and Bromus gros sus—influenced trait differentiation. Using plant material from the original source populations and from the ex situ collections, we compared germination characteristics, growth and phenology under different environmental treatments. Trifolium spadiceum showed reduced seed dormancy in the ex situ collection compared to the wild population, whereas germination temperature requirements changed for S. austriacum . Trifolium spadiceum also showed reduced seed viability in the ex situ collection compared to the wild population. All species showed differences in plant growth between the plants from nature and from the botanic garden. Additionally, B. grossus showed advanced flowering time in plants from the botanic garden. These differences may reflect reduced performance or changes in life-history strategies. We conclude that all three species have rapidly differentiated between wild and ex situ origins and that effects of relaxed selection, genetic drift, inbreeding depression and adaptation to cultivation conditions in the botanic garden may have played a role in population differentiation, which may be unfavourable for reintroduction into nature. To explore this further we suggest broader studies across more species, populations and gardens, involving common garden, reciprocal transplant and molecular studies.