Biological indicators, particularly benthic macroinvertebrates, are widely used and effective measures of the impact of urbanization on stream ecosystems. A multimetric biological index of urbanization was developed using a large benthic macroinvertebrate dataset (n = 1,835) from the Baltimore, Maryland, metropolitan area and then validated with datasets from Cleveland, Ohio (n = 79); San Jose, California (n = 85); and a different subset of the Baltimore data (n = 85). The biological metrics used to develop the multimetric index were selected using several criteria and were required to represent ecological attributes of macroinvertebrate assemblages including taxonomic composition and richness (number of taxa in the insect orders of Ephemeroptera, Plecoptera, and Trichoptera), functional feeding group (number of taxa designated as filterers), and habit (percent of individuals which cling to the substrate). Quantile regression was used to select metrics and characterize the relationship between the final biological index and an urban gradient (composed of population density, road density, and urban land use). Although more complex biological indices exist, this simplified multimetric index showed a consistent relationship between biological indicators and urban conditions (as measured by quantile regression) in three climatic regions of the United States and can serve as an assessment tool for environmental managers to prioritize urban stream sites for restoration and protection.