Dairy cattle are known reservoirs of pathogenic Escherichia coli, but little is known about the dynamics of E. coli in dairy cows or within the dairy farm environment. This study was conducted to evaluate the diversity and distribution of E. coli strains in a dairy farm using pulsed-field gel electrophoresis and to determine the relationships between E. coli isolated from feces and throughout the farm environment. Water from watering troughs, feces from cows, manure composites, milk, and milk filters were collected on December 2005 and December 2006. Isolates were analyzed by PCR for phylogenetic grouping (A, B1, B2, and D) and for the presence of virulence genes associated with enteropathogenic E. coli and enterohemorrhagic E. coli strains. Most of the isolates were in groups A (22%) and B1 (64%), while 4% and 11% of the isolates were within groups B2 and D, respectively. Enterohemorrhagic E. coli and enteropathogenic E. coli virulence genes were detected in strains from the feces of three cows and in one manure composite, and E. coli O157:H7 was present in one manure composite. Pulsed-field gel electrophoresis analysis resulted in 155 unique restriction digestion patterns (RDPs) among 570 isolates. E. coli isolates from water, manure composites, feces, milk, and milk filters grouped into 34, 65, 76, 4, and 6 clusters (identical RDPs), respectively. There was little diversity of isolates within individual fecal samples; however, high diversity was observed between fecal samples. Diversity was high within the water and composite samples. Some RDPs were common to multiple sample types. Although there were common RDPs between the 2005 and 2006 samplings, the E. coli populations were quite distinct between these two sampling times. These results demonstrate a high degree of diversity for E. coli within a dairy farm and that assigning a single environmental isolate to a particular farming operation would require the testing of an impractical number of isolates.