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In the alluvial valley of the lower Mississippi River, floodplain lakes form isolated aquatic fragments that retain differing degrees of connectivity to neighbouring rivers. Within these floodplain lakes it was hypothesized that fish species composition, relative abundance, and biodiversity metrics would be shaped largely by aquatic connectivity within a catchment.Fish assemblages in 13 floodplain lakes (five on‐channel; eight off‐channel) were assessed with electrofishing in 2006–2012 in the Bear Creek catchment, Mississippi, USA. Bear Creek spans approximately 80 km before draining into the Yazoo River, a tributary of the Mississippi River.Fish assemblages in on‐channel and off‐channel lakes were different, and fish assemblages in on‐channel lakes were as a group more homogeneous than off‐channel lakes. Moreover, a longitudinal gradient in fish assemblages occurred in on‐channel lakes. The observed patterns in fish assemblages are linked largely to differing intensities in connectivity among lakes within the catchment. Lakes with irregular connections have greater individuality, whereas lakes with continuous or chronic connection are more similar. The wide variation in connectivity could be a key to the distinctive biodiversity of catchments and the focus of fish conservation programmes.Off‐channel floodplain lakes are among the first landscape elements to vanish as a consequence of agricultural development. These habitats tend to accumulate sediments at fast rates and are converted to agricultural land as soon as suitable drainage can be attained. Considering that off‐channel lakes with limited connectivity contribute greatly to the heterogeneity of fish assemblages, such losses pose great concerns to conservation of biodiversity. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.