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Southern Brazilian rivers and streams have been intensively affected by human activities, especially agriculture and the release of untreated domestic sewage. However, data about the aquatic macroinvertebrates in these streams are scarce and limited to only certain groups. In addition, studies focusing on the structure and spatial distribution of these communities are lacking. This study analyzed the effects of natural and anthropic variables on the community structure of macroinvertebrates along a longitudinal gradient in three microbasins located in a region of landscape transition in the state of Rio Grande do Sul, Brazil. Sampling was conducted in the Vacacaí-Mirim River (August 2008) and in the Ibicuí-Mirim and Tororaipí rivers (August 2009) following an environmental gradient including 1st, 2nd, 3rd, and 4th order segments. Local natural factors that were analyzed include water temperature, pH, electrical conductivity, dissolved oxygen, substrate granulometry, and the presence of aquatic vegetation. Anthropic variables that were analyzed include including bank erosion, land use, urbanization, riparian deforestation, and fine sediments input. A total of 42 families and 129 taxa were found, with predominance of environmentally tolerant taxa. Geological context (landscape transition and large hydrographic basins) tended to influence natural environmental factors along the rivers' longitudinal gradients. However, changes in anthropic variables were not affected by these geological differences and therefore did not correlate with patterns of spatial distribution in macroinvertebrate communities. Only 1st order stream segments showed a community composition with high richness of taxa intolerant to anthropic disturbance. Richness as a whole tended to be higher in 3rd to 4th order set of segments, but this trend was a result of local anthropic environmental disturbances. Future inventories conducted in similar landscape transition regions of Brazil, for conservation purposes, must consider stream segments of different orders, microbasins, and major basins in order to obtain data that faithfully reflect the regional diversity. Additionally, it is necessary to consider environmental gradients of land use and anthropic impacts in order to suggest appropriate strategies for conserving the environmental integrity of streams.