We investigated the prevalence and diversity of Escherichia coli strains isolated from surface waters from multiple watersheds within the South Nation River basin in eastern Ontario, Canada. The basin is composed of mixed but primarily agricultural land uses. From March 2004 to November 2007, a total of 2,004 surface water samples were collected from 24 sampling sites. E. coli densities ranged from undetectable to 1.64 ؋ 10 5 CFU 100 ml ؊1 and were correlated with stream order and proximity to livestock production systems. The diversity of 21,307 E. coli isolates was characterized using repetitive extragenic palindromic PCR (rep-PCR), allowing for the identification of as many as 7,325 distinct genotypes, without capturing all of the diversity. The community was temporally and spatially dominated by a few dominant genotypes (clusters of more than 500 isolates) and several genotypes of intermediary abundance (clustering between 10 and 499 isolates). Simpson diversity indices, assessed on a normalized number of isolates per sample, ranged from 0.050 to 0.668. Simpson indices could be statistically discriminated on the basis of year and stream order, but land use, discharge, weather, and water physical-chemical properties were not statistically important discriminators. The detection of Campylobacter species was associated with statistically lower Simpson indices (greater diversity; P < 0.05). Waterborne E. coli isolates from genotypes of dominant and intermediary abundance were clustered with isolates obtained from fecal samples collected in the study area over the same period, and 90% of the isolates tested proved to share genotypes with fecal isolates. Overall, our data indicated that the densities and distribution of E. coli in these mixed-use watersheds were linked to stream order and livestock-based land uses. Waterborne E. coli populations that were distinct from fecal isolates were detected and, on this basis, were possibly naturalized E. coli strains.Escherichia coli is ubiquitously distributed in fecal material from humans and warm-blooded animals (38). The detection of E. coli in water is an implicit indicator of recent fecal contamination and therefore of the risk of cooccurrence of enteric pathogens that can cause illness in susceptible populations (62). Many jurisdictions evaluate and mandate compliance with drinking and recreational water quality standards on the basis of the presence and abundance of E. coli (14,44). For example, Canadian recreational water quality standards stipulate that E. coli densities in excess of a geometric mean of 200 CFU per 100 ml indicate that the water is unsuitable for swimming and bathing (23).In a background of increasing occurrence of microbial contamination of surface water, a variety of methods for elucidating the sources of fecal contamination have been developed, and these microbial source tracking (MST) methods are recommended components of fecal pollution abatement strategies (16,57). So-called library-dependent MST methods compare environmental isolates to co...