The catfish family Diplomystidae is one of the earliest branching lineages within the diverse order Siluriformes and shows a deep phylogenetic split from all other extant and extinct major catfish groups. Despite its relevance in the evolution of siluriforms, phylogenetic relationships within the Diplomystidae are poorly understood, and prior to this study, no phylogenetic hypotheses using molecular data had been published. By conducting a phylogeographic study across the entire distribution of the family, that encompasses river systems from Central-South Chile and Argentina, we provide the first molecular phylogenetic hypothesis among all known species of Diplomystidae, and in addition, investigate how their evolutionary history relates to major historical events that took place in southern South America. Our phylogenetic analyses show four main lineages and nine sub-lineages strongly structured geographically. All Pacific basin populations, with one exception (those found in the Baker basin) clustered within three of the four main lineages (clades I–III), while all populations from Atlantic basins and those from the Baker basin clustered in a single main clade (clade IV). There was a tendency for genetic diversity to decrease from north to south for Pacific basins consistent with an increasing north-south ice coverage during the last glacial maximum. However, we did not find a statistically significant correlation between genetic diversity and latitude. Analysis of molecular variance (AMOVA) showed that river basins and the barrier created by the Andes Mountains explained a high percentage of the genetic variation. Interestingly, most of the genetic variation among drainages was explained among Pacific basins. Molecular phylogenetic analyses agree only partially with current systematics. The geographical distribution of main lineages did not match species distribution and suggests a new taxonomic hypothesis with support for four species of Diplomystes, three species distributed allopatrically from the Rapel to the Valdivia basin, and only one species distributed in Baker and Atlantic basins. High genetic differentiation among river basins suggests that conservation efforts should focus on protecting populations in each basin in order to preserve the genetic diversity of one of the oldest groups of catfishes on the earth today.