Surveys performed in streams from the sub-basins of the Canoas and Pelotas rivers, which jointly form the Uruguay River in southern Brazil, led to the discovering of three new species of Aeglidae: Aegla brevipalma, Aegla leachi, and A. oblata. Here we present their morphological descriptions coupled with phylogenetic analyses, to infer evolutionary relationships to other aeglids using DNA sequence data from the 16S rRNA and COII gene regions of the mitochondrial genome. The presence of these new species increases the importance of the Upper Uruguay ecoregion for biodiversity richness of freshwater fauna in the Neotropical South America.