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The potential for mixtures of plant species to produce more biomass than every one of their constituent species in monoculture is still controversially discussed in the literature. Here we tested how this so-called transgressive overyielding is affected by variation between and within species in monoculture yields in biodiversity experiments.We use basic statistical principles to calculate expected maximum monoculture yield in a species pool used for a biodiversity experiment. Using a real example we show how between- and within-species variance components in monoculture yields can be obtained. Combining the two components we estimate the importance of sampling bias in transgressive overyielding analysis.The net biodiversity effect (difference between mixture and average monoculture yield) needed to achieve transgressive overyielding increases with the number of species in a mixture and with the variation between constituent species in monoculture yields. If there is no significant variation between species, transgressive overyielding should not be calculated using the best monoculture, because in this case the difference between this species and the other species could exclusively reflect a sampling bias. The sampling bias decreases with increasing variation between species. Tests for transgressive overyielding require replicated species’ monocultures. However, it can be doubted whether such an emphasis on monocultures in biodiversity experiments is justified if an analysis of transgressive overyielding is not the major goal.