Abstract The colonization pattern of fish assemblages in streams is often studied in the context of environmental filters. On the other hand, when fish assemblages are subjected to anthropogenic effects, variables associated with environmental quality assume more importance. Therefore, this work evaluated the richness and composition of fish from streams sampled at different urbanization levels, aiming to determine any direct effects on the structure of fish assemblages. To accomplish this, samples were collected from 2003 to 2011 at 31 sites distributed among 3 microbasins in the Rio Ivinhema Basin, Alto Rio Paraná. Based on environmental variables, physicochemical of the water and analysis of the use and occupation of the soil, the microbasins were classified into different urbanization levels (low, medium and high). A total of 4,320 individuals were sampled, out of which 57 fish species were recorded. Sampled sites with medium urbanization level presented higher richness compared to sampled sites with high urbanization level which presented lower richness. Species richness in these sites was explained mainly by water temperature and water velocity. Results confirmed that urbanization does directly affect environmental integrity, which, in turn, can lead to the homogenization of stream assemblages.