The complete D-loop sequence was obtained from 23 Hypostomus and 12 other species belonging to eight genera, from which seven are closely related to Hypostomus. The phylogenetic analysis of these sequences indicates that the genus Hypostomus is polyphyletic due to the exclusion of H. emarginatus and H. sgualinus from the main Hypostomus clade. Moreover, this main Hypostomus clade includes all Cochliodon species examined here, indicating that Cochliodon can be considered as a subjective junior synonym of Hypostomus. This conclusion is also supported by the placement of Hypostomus fonchii within the Cochliodon clade, implying that the synapomorphic characters diagnosing Cochhodon are unfounded. Based on these results, a new vision of Hypostomus is proposed. Furthermore, our results confirm the previously reported polyphyly of Ancistrinae and question the monophyletic status of Hemiancistrus.