Changes in land cover and fish assemblage structure were assessed across two spatial and temporal scales in the Piedmont and Coastal Plain regions of the Chesapeake Bay watershed in Virginia. A long-term, local study (1953 to 2014) on the Tuckahoe Creek watershed used digitized aerial photography and satellite images (Landsat 5 TM and Landsat 8 OLI/TIRS) to quantify land cover change for five nested catchments in 1953, 1990, and 2014. Instream fish collections from 1958, 1990, and 2014 were utilized to assess a variety of fish assemblage metrics for each of the five catchments, and analyses were performed to assess associations between changes in land cover and changes in fish assemblage structure across all three time periods. A short-term, regional study assessed 21 catchments in the region using 1997 Landsat 5 TM satellite images and 2014 Landsat 8 OLI/TIRS satellite images to quantify land cover change. Fish collections from 1995-1999 and 2014 were utilized to assess a variety of fish assemblage metrics from samples taken at instream sites for each of the 21 catchments. Analyses were performed to discover any associations between changes in land cover and changes in fish assemblage structure from a regional perspective. This study found that there were significant changes in land cover over all study periods in the Tuckahoe Creek watershed and that land cover changes were correlated to changes in fish assemblage structure over the long-term study. Regionally, there were significant changes in land cover, with no correlation to changes in fish assemblage structure found. The data suggests that anthropogenic alterations to the landscape have had long-term effects on fish assemblage structure in Tuckahoe Creek, but the results from the short-term assessments did not detect a relationship between land cover changes and changes in fish assemblage structure. It is possible that the fish communities were already established in moderately degraded catchments by the 1990s due to previous anthropogenic stressors.