We investigated the prevalence and diversity of
Escherichia colistrains 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. colidensities 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. coliisolates 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 Campylobacterspecies was associated with statistically lower Simpson indices (greater diversity; P< 0.05). Waterborne E. coliisolates 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. coliin these mixed-use watersheds were linked to stream order and livestock-based land uses. Waterborne E. colipopulations that were distinct from fecal isolates were detected and, on this basis, were possibly naturalized E. colistrains.