The lower Central American landscape was fully emergent approximately three million years ago, an event which marked the beginning of the Great American biotic interchange. Freshwater fishes participated in the biotic interchange. Because primary freshwater fishes are restricted to freshwater, they provide an excellent system for investigating the interplay of historical and recent processes on the assembly, structure and diversity of the region's aquatic ecosystems. We focused on examining the history of diversification for a species of catfish (Pimelodella chagresi) whose distribution spans multiple, isolated drainage basins across the Isthmian landscape and into north–western South America. Analysis of mitochondrial DNA haplotypes and morphological traits indicated that P . chagresi, as currently recognized, comprises a species complex. In addition, along the Pacific slope of Panama, repeated dispersion, diversification, extinction and possibly hybridization are thought to underlie a complex distribution of haplotypes. Overall, the results underscore the tremendous importance of historical processes on regional biodiversity.