We conducted this study to assess the influence of changes in freshwater inflow on the distribution and relative abundance of estuarine species in Rookery Bay, Florida. Originally, freshwater entered the bay via sheet-flow. In the 1960s, the creation of a canal system, which funnels water through a weir situated on Henderson Creek, altered inflows. To assess the effect of these changes, we initiated monitoring in Rookery Bay and Fakahatchee Bay. The latter estuary served as a control, because it still has natural sheet-flow.\Bottom types and bathymetry in Rookery Bay were mapped using side-scan and QTC View sonar systems. Bottom types were verified via analyses of bottom-grab and core samples. Salinity, temperature, and dissolved oxygen data collected seasonally in the two estuaries were interpolated to map water-column habitats.\When this study was initiated, no long-term fisheries-independent monitoring (FIM) had been conducted in Rookery Bay. Consequently, we analyzed FIM data collected in Charlotte Harbor to determine habitat affinities for 22 life stages of 11 species of fish and macroinvertebrates. Suitability functions were derived by fitting splines to catch rates (CPUEs) across environmental gradients and by calculating mean CPUEs within bottom-type categories. Abundance indices transferred from Charlotte Harbor were applied to Rookery Bay habitat layers so that we could conduct raster-based habitat suitability modeling (HSM) for a dry season (spring) and a wet season (summer) in Rookery Bay with differing freshwater inflows. Predicted HSM maps depict suitability zones (low to optimum) across the estuary. The models were validated by calculating mean observed CPUEs (from monthly trawl sampling) within the predicted zones. Increasing mean observed CPUEs across the zones indicated that the models adequately predicted the spatial distributions of the species’ life stages.