Populations of Escherichia coli from juvenile and adult ring-billed gulls, juvenile common terns, and adult Canada geese were sampled over 6 years at five locations on Lake Superior (Duluth, MN, and Wisconsin) and Lake Michigan (Wisconsin, Illinois, and Indiana) to determine the extent of spatial and temporal variability in E. coli strains. Strain identity was determined using horizontal fluorophore-enhanced repetitive element palindromic DNA fingerprinting. Multivariate statistics were used to determine if spatial or temporal changes in E. coli populations occurred in waterfowl species. Pairwise multivariate analyses of variance revealed that E. coli populations of adult gulls from three regions of Lake Michigan and the Wisconsin shore of Lake Superior were similar to one another but different from an E. coli population of gulls from the Duluth region of Lake Superior. Juvenile and adult gulls from the Duluth area harbored different E. coli populations. The E. coli strains from juvenile gulls, however, were similar to those found in juvenile terns obtained from the same island rookery. Temporal changes in E. coli populations from several waterfowl species were also demonstrated for this site. Although portions of source tracking databases might be successfully used in other geographic regions, it is clear that juvenile birds should not be the sole source of E. coli strains used for source tracking databases, and multiple-year libraries should be constructed in order to identify the potential sources of E. coli in the environment.