Many local and regional variables structure fish assemblage composition, although few studies have assessed the effects of aquatic connectivity on fish assemblages in wetlands. Fish and habitat surveys were conducted in 12 wetlands across the lower Great Lakes basin. Spatial and temporal connectivity was classified into four connectivity classes to evaluate the interaction between aquatic connectivity and fish assemblage structure. No significant relationship was identified between species richness and various area measurements. Species richness declined with decreased levels of connectivity, although no relationship was noted for abundance or diversity. A difference in piscivore richness was noted between wetland connectivity classes. Redundancy analysis (RDA) was used to examine the role of aquatic connectivity in structuring fish assemblages, and indicated that a combination of local environmental and area variables, in addition to aquatic connectivity, was best suited to describe the fish assemblage.