Ephemeroptera and Trichoptera larvae are important components of freshwater benthic communities. However, the environmental factors that determine Ephemeroptera and Trichoptera diversity and distribution in wetland systems of the Neotropical region are not well known. The objectives of this study were to: 1) conduct a diversity survey of mayflies and caddisflies in 140 wetlands in an extensive area of the Neotropical region (∼220,000 km2, southern Brazil), and 2) determine how much variation in mayfly and caddisfly richness and composition is explained by wetland area, altitude, hydroperiod, aquatic vegetation and water conductivity. A total of 5207 individuals distributed among 16 genera of Trichoptera and Ephemeroptera were collected in the studied wetlands. The mayflies were represented by eight genera, and comprised 94.6% of the collected individuals. The richness of Ephemeroptera and Trichoptera was similar between permanent and intermittent wetlands, and it did not differ significantly between aquatic bed and emergent wetlands. However, the density was higher in permanent than in intermittent wetlands, and it was higher in aquatic bed than in emergent wetlands. While the richness and density were negatively associated with altitude and water conductivity, the composition was associated with wetland area and altitude. In terms of the conservation, there is a need to promote conservation of all wetland systems in southern Brazil, regardless of their area and altitude. These arguments are essential to develop conservation and management programs of wetlands in this region.