Despite the fundamental importance of the family Diplomystidae for understanding catfish evolution, its species are poorly known and most of them endangered. Diplomystes camposensis, restricted to a single river basin in southern Chile, is perhaps the most vulnerable species due to its small geographic range and imminent habitat alterations by dam constructions. Using mitochondrial DNA sequences, we describe the genetic diversity across its entire distribution in the Valdivia basin and test hypotheses related to the impact of glacial cycles on the genetic diversity and structure. We found that Diplomystes camposensis has low genetic diversity and structure across the entire Valdivia basin along with a pattern of decreasing nucleotide and haplotype diversity from West to East. Demographic analyses showed evidence of population expansion in agreement with the glaciated history of the basin. Analyses of population structure showed no evidence of population subdivision. However, coalescent analyses indicated that very recent subdivision (in the last 50 years) cannot be ruled out. Low genetic diversity and genetic structure across the entire basin suggest that the species might be highly vulnerable to habitat fragmentation. Thus, the imminent construction of hydropower dams represents a serious threat to its conservation. Our results suggest that the low genetic diversity can be the product of the glaciated history of the basin, although the influence of species-specific biological traits may also add to this condition. Despite the overall low genetic diversity, higher diversity was found in the central portion of the basin suggesting high priority of conservation for this area as it might be used as a source population in case translocations are required among potential management plans.