The family Aspredinidae comprises a clade of complex systematic relationships, both from molecular and morphological approaches. In this study, conventional and molecular cytogenetic studies coupled with nucleotide sequencing were performed in 6 Aspredininae species (Amaralia hypsiura, Bunocephalus cf. aloikae, Bunocephalus amaurus, Bunocephalus aff. coracoideus, Bunocephalus verrucosus, and Platystacus cotylephorus) from different locations of the Amazon hydrographic basin. Our results showed highly divergent diploid numbers (2n) among the species, ranging from 49 to 74, including the occurrence of an XX/X0 sex chromosome system. A neighbor-joining phylogram based on the cytochrome c oxidase I (COI) showed that Bunocephalus coracoideus is not a monophyletic clade, but closely related to B. verrucosus. The karyotypic data associated with COI suggest an ancestral karyotype for Aspredinidae with a reduced 2n, composed of bi-armed chromosomes and a trend toward chromosomal fissions resulting in higher diploid number karyotypes, mainly composed of acrocentric chromosomes. Evolutionary relationships were discussed under a phylogenetic context with related species from different Siluriformes families. The karyotype features and chromosomal diversity of Aspredinidae show an amazing differentiation, making this family a remarkable model for investigating the evolutionary dynamics in siluriforms as well as in fish as a whole.