Oil spills might lead to severe environmental impacts to the affected fauna, disrupting local food webs, and causing mass mortality in many species. However, little is known about long-term impacts of oil spills, or even if such impacts can be detectable after several generations. In this study, we investigate the genetic variability of three freshwater species—Mimagoniates microlepis (Characiformes: Characidae), Scleromystax barbatus (Siluriformes: Callichthyidae), and Phalloceros harpagos (Cyprinodontiformes: Poeciliidae)—in rivers that were affected by a large oil spill in the state of Paraná, southern Brazil, on February of 2001. Samples were obtained from nine different locations, such that rivers that were directly affected by the oil spill could be compared with similar rivers in the same region that were unaffected. A fragment of the cytochrome C oxidase subunit I mitochondrial gene was sequenced from each specimen, and the level of genetic variability was assessed. Based on estimates of haplotype and nucleotide diversity, no impact of the oil spill could be detected in impacted rivers. These results suggest that fish populations in the region showed resilience to the pollutant, such that immigration from other locations was able to reestablish levels of genetic variability comparable to those of unimpacted rivers.