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Aim To provide a mechanistic and probabilistic framework for defining the species pool based on species-specific probabilities of dispersal, environmental suitability and biotic interactions within a specific temporal extent, and to show how probabilistic species pools can help disentangle the geographical structure of different community assembly processes. Innovation Probabilistic species pools provide an improved species pool definition based on probabilities in conjunction with the associated species list, which explicitly recognize the indeterminate nature of species pool membership for a given focal unit of interest and better capture real-world complexity. Probabilistic species pools provide a quantitative assessment of how dispersal, environmental or biotic factors influence estimates of species pool composition and size for a given temporal extent. Conclusions Based on one simulated and two empirical examples we demonstrate that probabilistic species pools allow us to disentangle the geographical variation in dispersal, environmental and biotic assembly processes for species assemblages in focal units. We also show that probabilistic species pools are fully compatible with traditional definitions of species pools and are applicable over a wide range of spatial and temporal extents. Additionally they are robust to missing data and provide a quantified and transparent approach to estimating the size and composition of species pools in a mechanistic way, providing a valuable tool for studies from community ecology to macroecology. [ABSTRACT FROM AUTHOR]