<p>Urbanization-induced environmental changes have become a major threat to river systems. However, little is known about how urbanization affects aquatic microbial communities. Here, the results highlight that nitrogen compounds can affect the microbial community composition. Proteobacteria and Bacteroidetes, as well as Cyanobacteria, were identified as the dominant phyla in urbanized rivers. Both the total diversity and total species richness of generalists were significantly higher than those of specialists, whereas the beta diversity of generalists was significantly higher in low-level urbanization sites (LUSs) than in high-level urbanization sites (HUSs). For generalists, heterogeneous selection is the most important process for the assembly of HUSs in winter, but dispersal limitation, drift, and other undominated processes are the main assembly processes in LUSs, whether in summer or winter. Similarly, stochastic processes were the main assembly of specialists in LUSs and HUSs, whether in summer or winter. A more complex network of generalists and specialists was found in the summer in HUSs and winter in LUSs, whereas a significantly stronger, more complex and robust co-occurrence network was found in the LUSs than in the HUSs. Overall, these findings suggest that specialists and nitrogen are key factors shaping the homogenization of microbial communities induced by urbanization.</p>