We surveyed fish assemblages from Blackwater River drainage (Cambridge, Maryland, USA) to assess spatiotemporal patterns of fish distribution in response to salinity intrusion of freshwater wetlands. Three sites spaced along a longitudinal stream gradient were sampled from each of the Little Blackwater River and Blackwater River from December 2005 to December 2006. Salinity in upstream areas of Blackwater River ranged from 9–12 psu, while in the Little Blackwater River salinity ranged from 0–5 psu. Most of the spatial differences in assemblage structure occurred between upstream sites of Blackwater River, an area influenced by saltwater intrusion from the combined effects of sea level rise and a man-made canal, and upstream sites of Little Blackwater River. Freshwater-dependent fishes (e.g., brown bullhead and black crappie) dominated upstream sites of Little Blackwater River, whereas euryhaline species (e.g., killifish and silversides) dominated upstream sites of Blackwater River. We used the Little Blackwater River as a reference system to test the hypothesis that remnant freshwater-dependent fish populations of transitional areas in the Blackwater River may have declined as salinity levels increased. Upstream habitats of Blackwater River were brackish, while such habitats of Little Blackwater River were more persistently freshwater. As salinity increased seasonally in the Little Blackwater River, the abundance of freshwater-dependent fishes declined at two sites. Differences in species diversity and composition between Blackwater River and Little Blackwater River are likely associated with higher salinity and connectivity in upstream portions of the Blackwater River.