Ecoregions and areas of endemism are central concepts in biogeography. Based on collection records and the Endemic Analyses implemented with NDM/VNDM method we analyzed fish areas of endemism in the junction of three freshwater ecoregions related to the Rio de la Plata estuary (Lower Parana, Lower Uruguay, Laguna dos Patos) in Southern Neotropical region. Using two grid cell sizes, results obtained showed the same general patterns. Areas of endemism recovered were mainly associated either to Lower Uruguay or to Laguna dos Patos. In both ecoregions nested areas of endemism were identified within larger patterns of endemism. Noteworthy, one area recovered occurred across Lower Uruguay and Laguna dos Patos limits. Our results also suggest a revision of the Lower Uruguay and Lower Parana ecoregion limits, and highlight the relevance of the Rio de la Plata estuary as a barrier and corridor for freshwater fishes in the area.