Aim: To study the diversity of commensal Escherichia coli populations shed in faeces of cattle fed on different diets. Methods and Results: Thirty Brahman-cross steers were initially fed a high grain (80%) diet and then randomly allocated into three dietary treatment groups, fed 80% grain, roughage, or roughage + 50% molasses. Up to eight different E. coli isolates were selected from primary isolation plates of faecal samples from each animal. Fifty-two distinct serotypes, including nine different VTEC strains, were identified from a total of 474 E. coli isolates. Cattle fed a roughage + molasses diet had greater serotype diversity (30 serotypes identified) than cattle fed roughage or grain (21 and 17 serotypes identified respectively). Cluster analysis showed that serotypes isolated from cattle fed roughage and roughage + molasses diets were more closely associated than serotypes isolated from cattle fed grain. Resistance to one or more of 11 antimicrobial agents was detected among isolates from 20 different serotypes. Whilst only 2.3% of E. coli isolates produced enterohaemolysin, 25% were found to produce alpha-haemolysin. Conclusions: Diverse non-VTEC populations of E. coli serotypes are shed in cattle faeces and diet may affect population diversity. Significance and Impact of the Study: This study provides new information on the serotype diversity and phenotypic traits of predominant E. coli populations in cattle faeces, which could be sources of environmental contamination.