We examined associations of fish assemblages and fish traits with urbanization and selected environmental variables in nine major United States metropolitan areas. The strongest relations between fishes and urbanization occurred in the metropolitan areas of Atlanta, Georgia; Birmingham, Alabama; Boston, Massachusetts; and Portland, Oregon. In these areas, environmental variables with strong associations (rs ≥ 0.70) with fish assemblages and fish traits tended to have strong associations with urbanization. Relations of urbanization with fish assemblages and fish traits were weaker in Denver, Colorado; Dallas-Fort Worth, Texas; Milwaukee-Green Bay, Wisconsin; and Raleigh, North Carolina. Environmental variables associated with fishes varied among the metropolitan areas. The metropolitan areas with poor relations may have had a limited range of possible response because of previous landscape disturbances. Given the complexities of urban landscapes in different metropolitan areas, our results indicate that caution is warranted when generalizing about biological responses to urbanization.