ABSTRACT Catfishes of the family Astroblepidae form a group composed by 82 valid species of the genus Astroblepus inhabiting high-gradient streams and rivers throughout tropical portions of the Andean Cordillera. Little has been advanced in the systematics and biodiversity of astroblepids other than an unpublished thesis, a single regional multilocus study and isolated species descriptions. Here, we examined 208 specimens of Astroblepus that apparently belong to 16 valid species from several piedmont rivers from northern Colombia to southern Peru. Using three single-locus approaches for species delimitation in combination with a species tree analysis estimated from three mitochondrial genes, we identified a total of 25 well-delimited lineages including eight valid and 17 potential undescribed species distributed in two monophyletic groups: the Central Andes Clade, which contains 14 lineages from piedmont rivers of the Peruvian Amazon, and the Northern Andes Clade with 11 lineages from trans- and cis-Andean rivers of Colombia and Ecuador, including the Orinoco, Amazon, and Magdalena-Cauca basins and Pacific coastal drainages. Results of species delimitation methods highlight several taxonomical incongruences in recently described species denoting potential synonymies.