While genotypically-distinct naturalized Escherichia coli strains have been shown to occur in riparian soils of Lake Michigan and Lake Superior watersheds, comparative analyses of E. coli populations in diverse soils across a range of geographic and climatic conditions have not been investigated. The main objectives of this study were to: (a) examine the population structure and genetic relatedness of E. coli isolates collected from different soil types on a tropical island (Hawaii), and (b) determine if E. coli populations from Hawaii and temperate soils (Indiana, Minnesota) shared similar genotypes that may be reflective of biome-related soil conditions. DNA fingerprint and multivariate statistical analyses were used to examine the population structure and genotypic characteristics of the E. coli isolates. About 33% (98 of 293) of the E. coli from different soil types and locations on the island of Oahu, Hawaii, had unique DNA fingerprints, indicating that these bacteria were relatively diverse; the Shannon diversity index for the population was 4.03. Nearly 60% (171 of 293) of the E. coli isolates from Hawaii clustered into two major groups and the rest, with two or more isolates, fell into one of 22 smaller groups, or individual lineages. Multivariate analysis of variance of 89, 21, and 106 unique E. coli DNA fingerprints for Hawaii, Indiana, and Minnesota soils, respectively, showed that isolates formed tight cohesive groups, clustering mainly by location. However, there were several instances of clonal isolates being shared between geographically different locations. Thus, while nearly identical E. coli strains were shared between disparate climatologically- and geographically-distinct locations, a vast majority of the soil E. coli strains were genotypically diverse and were likely derived from separate lineages. This supports the hypothesis that these bacteria are not unique and multiple genotypes can readily adapt to become part of the soil autochthonous microflora.