We examined patterns in Maryland fish assemblages in 1^st- through 3^rd-order nontidal streams along an urbanization gradient in the Eastern Piedmont (EP) and Coastal Plain (CP) physiographic ecoregions of Maryland, USA, using 1995 to 1997 and 2000 to 2002 data from the Maryland Biological Site Survey (MBSS). Major urbanization and other historical stressors occur in both ecoregions, and there is potential for further stress over the next 25 y as urbanization increases. We assigned each MBSS site (n = 544 streams) to a class of urbanization based on land cover within its upsite catchment. We compared observed fish abundance and species richness to the probable (expected) assemblages within each ecoregion, and also assessed the accuracy of the Maryland fish index of biotic integrity (FIBI) to indicate catchment urbanization. Relationships between urbanization and fish assemblages and FIBI varied between the 2 ecoregions. Assemblages in EP streams exhibited stronger relationships with urbanization than those in CP streams, particularly when urban land cover was >25% of the catchment. Across all EP stream orders (1^st, 2^nd, and 3^rd), high urbanization was associated with low fish abundance and richness, low FIBI, and few intolerant fish species, resulting in assemblages dominated by tolerant species. Conservation practices minimizing urbanization effects on fish assemblages may be inadequate to protect sensitive fish species because of the invasiveness of urban development and stressors related to the urban stream syndrome.