Between May 2004 and May 2005, we sampled fish in 19 sites, grouped in four elevations, ranging from the river mouth to 650 m.a.s.l. in the Río Pacuare, Caribbean versant of Costa Rica. Changes in the distribution and composition of the fish fauna, as well as patterns of alpha and beta diversity along an elevational gradient were assessed. Additional analyses of habitat preferences, trophic guilds, functional groups and general ecology for the most abundant species are included. All fish captured were classified into 22 families, 43 genera and 53 species. The most abundant family was Characidae, followed by Gobiidae, Mugilidae, Poeciliidae and Heptateridae, which together comprise 87.9 % of all sampled individuals. Elevation shows an inverse effect on species diversity, we observed a monotonic decrease in species richness with increasing elevation (p < 0.05), as reported in other tropical rivers. According to our results, in the Río Pacuare the total fish fauna diversity is found within the first 500 m.a.s.l. Species turnover increases with elevation, while nestedness decreases. Turnover was dominated by the loss of species rather than gain; the higher species loss was registered between the river mouth and the lower river reach (< 100 m.a.s.l.). Seven species can be classified as typical or core species (Astyanax aeneus, Sicydium altum, Agonostomus monticola, Poecilia gillii, Brycon costaricensis, Rhamdia laticauda and Joturus pichardi) along the elevation gradient. The habitat availability and the integration of ecomorphological, feeding and reproductive traits help to explain better the elevation distribution of the complete set of species observed. Although it is possible to identify groups of species characteristic of each reach of river, this does not mean that they are isolated from each other. Natural drift and movement along the river of some species during their life cycle, especially S. altum, A. monticola and J. pichardi, are key processes linking the whole watershed. The present study constitutes a first step in documenting and understanding the distribution and composition of fish assemblages in a watershed that is relatively intact and well-conserved in the Caribbean versant of Costa Rica.