Abstract: A number of studies have pointed out that abiotic factors and recolonization dynamics appear to be more important than biotic interactions in structuring river–fish assemblages. In this paper, we studied the fish assemblages in 27 floodplain lakes, with perennial connection to the river, in the middle section of the Magdalena River (Colombia), to examine spatial pattern in freshwater fish diversity in relation to some environmental parameters. Our objective was to examine relationships between floodplain-lake fish communities and environmental variables associated with lake morphology, water chemistry and river–floodplain connectivity in a large river–floodplain ecosystem. During the study, a total of 18 237 fish were caught from 50 species (regional richness; 17 were migrants and 33 residents). In the present study, the most diverse order was Characiformes with 20 species, followed by Siluriformes, with 19 species. Characidae and Loricaridae were the richest families. The range of species richness (local richness) varied between five and 39 species. Similarity of local assemblages (using the presence–absence data) depends on the distance between lakes. A positive relationship was observed between the Ln of the total abundance of each species and the number of lakes where they were found. Out of all the environmental parameters taken in the lakes, only the size (Log Area) and relative perimeter length are significantly related to local assemblage species richness. It has not been possible to demonstrate that the connectivity (distance) from lakes to the main river can be considered a predictor of the local richness.