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Abstract Replacing native forests by alien tree plantations can lead to changes in the species composition of the understory. However, differences in the understory species spectrum can also be a part of the natural variability of forest stands. We have tested the suitability of the twin plots method for an evaluation of the impact of alien trees on the species composition of the understory. This research was conducted on an alluvial plain (SW Slovakia) that was originally covered by a hardwood floodplain forest. The study was based on 7 twin plots of black locust (Robinia pseudoacacia) and native forest plots, with a maximum distance of 100 meters between the members of the twins. The dissimilarity of the plots within the black locust forest was significantly lower than the dissimilarity between the twin plots. In addition, the dissimilarity of the plots within the hardwood floodplain forest was also significantly lower than the dissimilarity between the twin plots. Under the same environmental conditions, the higher dissimilarity of the twin plots was caused by major edificators and their impact on the understory vegetation. The twin plots method proved to be a suitable tool for analyses of the impact of alien trees on understory vegetation.