Freshwater microbial diversity is subject to multiple stressors in the Anthropocene epoch. However, the effects of climate changes and human activities on freshwater protozoa remain poorly understood. In this study, the diversity and distribution of testate amoebae from the surface sediments were investigated in 51 Chinese lakes and reservoirs along two gradients, latitude and trophic status. A total of 169 taxa belonging to 24 genera were identified, and the most diverse and dominant genera were Difflugia (78 taxa), Centropyxis (26 taxa) and Arcella (12 taxa). Our analysis revealed that biomass of testate amoebae decreased significantly along the latitudinal gradient, while Shannon-Wiener indices and species richness presented an opposite trend (P < 0.05). The relationship of diversity and latitude is, we suspect, an artifact of the altitudinal distribution of our sites. Furthermore, biomass-based Shannon-Wiener index and species richness of testate amoebae were significantly unimodally related to trophic status (P < 0.05). This is the first large-scale study showing the effects of latitude and trophic status on diversity and distribution of testate amoebae in China. Therefore, our results provide valuable baseline data on testate amoebae and contribute to lake management and our understanding of the large-scale global patterns in microorganism diversity.