An understanding of how genetic variability is distributed in space is fundamental for the conservation and maintenance of diversity in spatially fragmented and vulnerable populations. While fragmentation can occur from natural barriers it can also be exacerbated by anthropogenic activities such as hydroelectric power plant development. Whatever the source, fragmentation can have significant ecological effects, including the disruptions of migratory processes and gene flow among populations. In Chile, the Biobío river basin exhibits a high degree of habitat fragmentation due to the numerous hydroelectric power plants in operation, the number of which is expected to increase following new renewable energy use strategies. Here, we assessed the effects of different kinds of barriers on the genetic structure of the endemic freshwater fish Percilia irwini, knowledge that is critically needed to inform conservation strategies in light of current and anticipated further fragmentation initiatives in the system. We identified 8 genetic units throughout the entire Biobío system with high effective sizes. A reduced effective size estimate was however observed in a single population located between two impassable barriers. Both natural waterfalls and human made dams were important drivers of population differentiation in this system, however, dams affect genetic diversity differentially depending on their mode of operation. Evidence of population extirpation was found in two river stretches limited by upstream and downstream dams. Significant gene flow in both directions was found among populations not separated by natural or anthropogenic barriers. Our results suggest a significant vulnerability of P. irwini populations to future dam development and demonstrate the importance of studying basin-wide data sets with genetic metrics to understand the strength and direction of anthropogenic impacts on fish populations.