Phylogeography and molecular phylogenetics have proven remarkably useful for understanding the patterns and processes influencing historical diversification of biotic lineages at and below the species level, as well as delimiting morphologically cryptic species. In this dissertation, I used an integrative approach coupling comparative phylogeography and coalescent-based species delimitation to improve our understanding of the biogeography and species limits of Central American freshwater fishes. In Chapter 1, I conducted a literature review of the contributions of phylogeography to understanding the origins and maintenance of lower Central American biodiversity, in light of the geological and ecological setting. I highlighted emerging phylogeographic patterns, along with the need for improving regional historical biogeographical inference and conservation efforts through statistical and comparative phylogeographic studies. In Chapter 2, I compared mitochondrial phylogeographic patterns among three species of livebearing fishes (Poeciliidae) codistributed in the lower Nicaraguan depression and proximate uplands. I found evidence for mixed spatial and temporal divergences, indicating phylogeographic “pseudocongruence” suggesting that multiple evolutionary responses to historical processes have shaped population structuring of regional freshwater biota, possibly linked to recent community assembly and/or the effects of ecological differences among species on their responses to late Cenozoic environmental events. In Chapter 3, I used coalescent-based species tree and species delimitation analyses of a multilocus dataset to delimit species and infer their evolutionary relationships in the Poecilia sphenops species complex (Poeciliidae), a widespread but morphologically conserved group of fishes. Results indicated that diversity is underestimated and overestimated in different clades by c. ±15% (including candidate species); that lineages diversified since the Miocene; and that some evidence exists for a more probable role of hybridization, rather than incomplete lineage sorting, in shaping observed gene tree discordances. Last, in Chapter 4, I used a comparative phylogeographical analysis of eight codistributed species/genera of freshwater fishes to test for shared evolutionary responses predicted by four drainage-based hypotheses of Neotropical fish diversification. Integrating phylogeographic analyses with paleodistribution modeling revealed incongruent genetic structuring among lineages despite overlapping ancestral Pleistocene distributions, suggesting multiple routes to community assembly. Hypotheses tests using the latest approximate Bayesian computation model averaging methods also supported one pulse of diversification in two lineages diverged in the San Carlos River, but multiple divergences of three lineages across the Sixaola River basin, Costa Rica, correlated to Neogene sea level events and continental shelf width. Results supported complex biogeographical patterns illustrating how species responses to historical drainage-controlling processes have influenced Neotropical fish diversification.