Tropical South America possesses the largest ichthyofauna of any continental region. To test whether palaeohydrological changes may have been the causes of such diversification, the ‘hydrogeological’ hypothesis, the phylogenetic relationships of 51 representatives of the catfish genus Hypostomus (Siluriformes: Loricariidae) were inferred using mitochondrial D-loop haplotype sequences. Specimens were collected in all main tropical South American rivers systems east to the Andes. The major interrelationships found with the D-loop data were confirmed with a subset of 21 species using complete internal transcribed spacer (ITS) region sequences. The phylogenetic analysis indicate that the genus Hypostomus can be divided into four monophyletic clades. The historical biogeographical analysis of each of these clades allows the identification of seven major cladogenetic events. Using calibrated D-loop and ITS molecular clocks, date estimations were attributed to each of these cladogenetic events allowing a linkage between four of them with documented hydrogeological changes. Comparisons with published distribution patterns of unrelated fish groups indicate that several of the reconstructed and dated hydrogeological–cladogenetic events may have acted at a large scale on the diversification of Neotropical freshwater fish fauna during late Tertiary.