Abstract Aim This paper examines the importance of regional processes in determining the patterns of distribution and diversity of lower Mesoamerican freshwater fishes. Location We focused our analyses on the lower Mesoamerican region, which we define to include all the rivers of Panama and Costa Rica. The geographic boundaries are the Colombian Choco to the south and Lake Nicaragua to the north. Methods We described the biogeographical provinces of lower Mesoamerica (LMA) using presence/absence data of primary and secondary LMA freshwater fishes. We conducted subsequent analyses at the spatial resolution of the biogeographical provinces and described patterns of community composition, species richness, endemism, range size, and the permeability of dispersal barriers between biogeographical provinces. Results This study represents the first attempt since that of W. A. Bussing in 1976 to investigate the biogeographical regions of Mesoamerica, and our analyses demonstrate increased regional complexity in biodiversity patterns relative to previous studies. Changes in community composition across LMA clearly highlight the importance of both extrinsic geological processes and intrinsic biological differences among freshwater fish species in shaping the dispersal and diversification histories of the LMA freshwater fish fauna. The influence of biology and geology is also exemplified by patterns of endemism and turnover between biogeographical provinces, which suggests that the relative importance of regional speciation and dispersal varies spatially across the LMA landscape. Finally, it would seem to follow that secondary freshwater fishes will have larger range sizes than primary fishes as a result of the increased salinity tolerance posited for the former group, and thus the increased probability of dispersal along coastlines. We did not, however, find a significant difference between the average range size of primary and secondary freshwater fishes, indicating that the putative differences in physiological tolerance to seawater between the two groups are not reflected in their distribution patterns at the scale of LMA. The geometric distribution of range size of LMA freshwater fishes suggests that dispersal of both primary and secondary freshwater fishes along coastlines must be infrequent. Main conclusion The observation that regional processes exerted a strong influence on the assembly and maintenance of LMA freshwater fish communities has important consequences for both theory and conservation. We suggest that large-scale biogeographical analyses are required to illuminate the backdrop upon which local interactions play themselves out, supporting a top-down approach to the study of biological diversity. Our results also identify areas of high conservation priority, providing a baseline for informing conservation strategies for freshwater fishes in LMA. We conclude by calling for conservation planning and action that acknowledges the importance that regional processes play in determining patterns of organismal diversity, and that incorporates these processes in strategies to conserve remnant biological diversity.