A new species of Austrolebias is described based on individuals from the middle and upper río Negro (río Uruguay basin) and río Yaguarón (Patos-Merín system). The new species can be differentiated from all other species of the genus by the unique presence in males of uniform bluish gray pigmentation on flanks (without vertical bands) and unpaired fins. The new species is also distinguished by the combination of characters associated with a reduction of the squamation of the abdominal, preopercular, and opercular regions. The new species presents some morphological characteristics similar to A. gymnoventris and A. luteoflammulatus. The distribution of the new species is concordant with three other species of Austrolebias and may represent a case of drainage rearrangement of the río Negro upstream tributaries (río Uruguay basin) and tributaries of laguna Merín system