To evaluate the influence of main channel–floodplain connectivity on fish assemblage diversity in floodplains associated with streams and small rivers, fish assemblages and habitat characteristics were surveyed at 24 stream reaches in the Champlain Valley of Vermont, U.S.A. Fish assemblages differed markedly between the main channel and the floodplain. Fish assemblage diversity was greatest at reaches that exhibited high floodplain connectivity. Whereas certain species inhabited only main channels or floodplains, others utilized both main channel and floodplain habitats. Both floodplain fish α‐diversity and γ‐diversity of the entire stream corridor were positively correlated with connectivity between the main channel and its floodplain. Consistent with these results, species turnover (as measured by β‐diversity) was negatively correlated with floodplain connectivity. Floodplains with waterbodies characterized by a wide range of water depths and turbidity levels exhibited high fish diversity. The results suggest that by separating rivers from their floodplains, incision and subsequent channel widening will have detrimental effects on multiple aspects of fish assemblage diversity across the stream–floodplain ecosystem.