The family Loricariidae, with about 683 species, is one the largest fish families in the world. The subfamily Hypostominae was recently reviewed and is now divided in five tribes. With the main objective of contributing to a better understanding of the relationships of the members of the subfamily Hypostominae, cytogenetic analyses were conducted in seven species (three Hypostomini, three Pterygoplichthini and two Ancistrini) from Brazil and Venezuela. In Pterygoplichthini, all species show 2n = 52 chromosomes. In Hypostomini Hypostomus ancistroides has 2n = 68, H. regani 2n = 72 and Hypostomus goyazensis 2n = 72 chromosomes. In Ancistrini Ancistrus n. sp. 1 has 2n = 39/40 with a sex chromosome system of the type XX/X0, which is a novelty for neotropical fishes, and Ancistrus n. sp. 2 has 2n = 52 chromosomes. Six species have single Ag-NORs and two multiple Ag-NORs. The possible cytogenetic relationships among the species of Hypostominae are discussed.