Partial 12S and 16S mitochondrial rRNA gene sequences were obtained from 16 species of South American catfishes belonging to the subfamilies Hypostominae, Ancistrinae, and Loricariinae sensu Isbrücker (Siluroidei: Loricariidae). The analysis of these sequences indicates that within the clade of Hypostominae + Ancistrinae, Chaetostoma + Ancistrus form the sister group of all other analysed ancistrines and hypostomines. The Ancistrinae as presently defined, include all analysed Hypostominae and therefore are paraphyletic. The monophyly of Ancistrus, including the recently described A. ranunculus, is strongly supported.