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The richness and composition of a mountain-river chironomid larvae assemblage in the Jacuí River basin, Brazil were studied, and compared with other riverine non-biting midge larvae assemblages previously studied in the country. Additionally, the influence of some regional-scale environmental characteristics on the spatial distribution of these assemblages was tested. The specimens were collected at 12 sites in the middle course of the Jacuí River basin (in the state of Rio Grande do Sul) between April 2000 and May 2002. Around 100 taxa were recorded. The dominant taxa belonged to the genera Rheotanytarsus, Cricotopus, Polypedilum, and Pseudochironomus. Twenty-two rare taxa were found, representing 22% of the total of taxa inventoried. Fourteen genera (Aedokritus, Axarus, Endotribelos, Kiefferulus, Manoa, Oukuriella, Phaenopsectra, Stenochironomus, Xenochironomus, Xestochironomus, Cardiocladius, Metriocnemus, Paracladius, and Rheocricotopus) represent new occurrences in Rio Grande do Sul. The similarity analysis of the chironomid larvae assemblages inventoried in 32 regions of Brazil indicated five groups with similarity higher than 50%. The groups, when the effects of spatial autocorrelation were removed, displayed a weak positive correlation between the assemblage composition and the aquatic system or hydraulic conditions and the hydrographic basin, and a weak negative correlation in relation to the biome. The altitude showed no correlation with the composition of the assemblage. The relatively high richness of the region surveyed in relation to other Brazilian regions corroborates some tendencies already noted in other parts of the world, such as: i) lotic systems may constitute an exception to the rule that diversity is greater in tropical regions, ii) regions of transitional relief may contain the greatest richness of Chironomidae, and iii) in rivers, the group might have its spatial distribution influenced to a greater extent by local environmental characteristics than by regional ones.