Understanding the effects of urbanization on the diversity of freshwater macroinvertebrates is an important topic of biodiversity research and has direct conservation relevance. The absence of evidence-based systematic overviews on this topic motivated us to perform meta-analyses and to synthetize the present state of knowledge. We observed significant heterogeneity among individual case studies, reporting negative, neutral and positive effects. As expected, urbanization had an overall negative effect on the diversity of freshwater macroinvertebrates. These results are based mainly on the study of lotic (stream and river) ecosystems because there are insufficient data available for lentic (pond and lake) ecosystems. Compared to individual case studies, the present review reports an evidence-based synthesis for the first time. We identified knowledge gaps regarding case studies reporting the effects of urbanization on pond and lake ecosystems, case studies examining the phylogenetic and functional facets of biodiversity, as well case studies investigating the effect of urbanization on the beta diversity component of macroinvertebrate communities. The identification of these knowledge gaps allowed us to make recommendations for future research: (1) report results on specific taxonomic groups and not only the entire macroinvertebrate community, (2) study the impacts of urbanization on macroinvertebrate diversity in different habitat types and understudied continents, (3) focus on the functional and phylogenetic facets of diversity and (4) examine the influence of spatial scale on biodiversity (e.g. beta diversity) in urban freshwater ecosystems. Our results also suggested that the analysis of diversity-environment relationships is crucial for developing macroinvertebrate indicators especially in the increasingly urbanized world. Keywords aquatic invertebrates, biodiversity, effect of urbanization, freshwater ecosystems, systematic review 1. Introduction Sixty-eight percent of the global population is expected to live in cities by 2050, and the most urbanized regions are North America (with 82% of its population living in urban areas in 2018), Latin America and the Caribbean (81%), and Europe (74%). At the same time, individual cities are also growing in the developing world, resulting in new megacities (UNDESA, 2018). The proliferation of densely-settled areas from the coastal zone to the upstream regions, including mega-cities, means that many rivers are highly threatened over virtually their entire length (Vörösmarty et al., 2010). These freshwater systems have been modified throughout human history to serve humankind, including land cover change, urbanization and industrial purposes. In addition, we have been tireless advocates for expanding the access to the water for many uses and services. Because of the varied economic benefits of the water, it is a challenge to balance between societal and ecological needs (Geist and Hawkins, 2016). Urbanization alters the physical and chemical environment of rivers, streams...