Wetlands are important ecosystems in southern Brazil because they show high productivity and biological diversity. However, conservative data indicate that ~90% of the wetlands have disappeared as a result of agricultural expansion. In this sense, the understanding of species composition and richness patterns in fragmented and natural wetlands is a priority for biodiversity conservation strategies. The main goal of the present study was to determine how much variation in macroinvertebrate richness and composition is explained by wetland area, hydroperiod and altitude. This survey was carried out in an extensive area of the Neotropical region (~280 000 km² – southern Brazil) with a large number of wetland systems (72) and covering a wide gradient of altitude and wetland surface area. The macroinvertebrate richness was higher in permanent wetlands than intermittent ones, and intermittent wetlands supported a macroinvertebrate composition that clearly differed from those of permanent wetlands. Macroinvertebrate richness was positively affected by wetland area; however, this relationship was significant only in permanent wetlands. There is a need to promote conservation of all wetland systems in southern Brazil, regardless of their hydroperiod and area. These arguments are essential to develop conservation and management programs of wetlands in this region.