The Yazoo River Basin of Mississippi includes several rivers that drain an area heavily impacted by agriculture that includes hundreds of fluvial lakes created by the meandering of the rivers. We studied 17 of these lakes distributed over the lower half of the Yazoo River Basin to document fish assemblage patterns and identify environmental variables that might influence these assemblages. Results of multivariate analyses showed that the hydrological connectivity and wetland buffer played a major role on the limnological conditions and fish communities. Lakes with direct connections to the parent river and limited forested wetland buffers tend to be deeper, less turbid, lower phytoplankton fluorescence, and had greater fish species richness that included more riverine species. Conversely, as connectivity to the parent river is reduced and the presence of forested wetland buffers increase, lakes become shallow, turbid, and have higher phytoplankton fluorescence and a less speciose lacustrine fish community. These results suggested that the river connectivity could be an important factor in determining fish community and physicochemical conditions of these floodplain lakes.