Urbanization is restructuring ecosystems at an unprecedented pace, with complex and profound consequences for life on Earth. One of the hypothesized trajectories of urban ecosystems and species communities is biotic homogenization, possibly leading to very similar species assemblages in cities across the globe. Urbanization can, however, also have the opposite effect: biotic diversification, with cities, at least at the local scale, becoming biologically more diverse, mainly as a consequence of high species introduction rates and habitat diversification. Applying the hierarchy-of-hypotheses approach, we systematically map and structure the comprehensive body of literature on the urban biotic homogenization (UBH) hypothesis, comprising 225 individual studies (i.e., tests of the hypothesis) retrieved from 145 publications. The UBH hypothesis is studied at multiple levels with a multitude of approaches and underlying assumptions. We show that UBH is generally used with two very different connotations: about half of the studies investigated a potential increase in community similarity across cities, whereas the other half investigated biotic homogenization within cities, the latter being supported more frequently. We also found strong research biases: (1) a taxonomic bias towards birds and plants, (2) a bias towards small and medium distances (<5000 km) in comparisons across cities, (3) a dominance of studies substituting space for time versus true temporal studies, (4) a strong focus on terrestrial versus aquatic systems, (5) more extraurban (including periurban) areas than natural or rural ecosystems for comparison to urban systems, (6) a bias towards taxonomic versus functional, phylogenetic, and temporal homogenization, and (7) more studies undertaken in Europe and North America than in other continents. The overall level of empirical support for the UBH hypothesis was mixed, with 55% of the studies reporting supporting evidence. Results significantly differed when a natural/nature reserve, an extraurban, or rural/agricultural area served as reference to infer biotic homogenization, with homogenization being detected least frequently when urban systems were compared to agricultural, i.e., other anthropogenically influenced, study sites. We provide an evidence map and a bibliographic network and identify key references on UBH with the goal to enhance accessibility and orientation for future research on this topic.