Escherichia coli (E. coli) isolate diversity enhances the likelihood of survival, spread, and/or transmission of the organism among environments. Understanding the ecology of this important organism is requisite for development of more accurate protocols for monitoring and regulatory purposes. In this study, E. coli diversity, gene profiles and transport properties of isolates from different livestock and water sources were evaluated. Strain diversity was evaluated by BOX-PCR, phylotyping, and profiling for 15 genes associated with adhesion, toxin production, iron acquisition or capsular synthesis. Attachment efficiencies were calculated for 17 isolates following transport through saturated porous media. Richness of genotype profiles for livestock isolates was relatively low (25, 12, and 11 for swine, poultry and dairy, respectively) compared to those from stream-water (115 and 126 from dry or wet weather events, respectively). Attachment efficiencies varied by an order of magnitude (0.039-0.44) and the isolate with the highest attachment efficiency possessed the largest suite of targeted genes including those for adherence (iha, agn43, and fimH), surface exclusion (traT) and the siderophore iroN (E.coli) . Variation in E. coli isolates based on temporal and ecological source was found to translate to equally broad ranges in transport efficiency underscoring the large degree of genotypic and phenotypic variation that exists among E. coli isolates. The impact of this diversity on genetic exchange and the concomitant effect on the organisms' fate and transport under in situ environmental conditions warrant further investigation. These factors also require careful consideration for purposes of modeling, source tracking, and risk assessment.