Aims: The aim of this study was to assess geographical variation in multiple antibiotic resistance (MAR) profiles of livestock Escherichia coli as well as to evaluate the ability of MAR profiles to differentiate sources of faecal pollution. Methods and Results: More than 2000 E. coli isolates were collected from water retention ponds and manure of swine, poultry, beef and dairy farms in south, central and north Florida, and analysed for MAR using nine antibiotics. There were significant differences in antibiotic resistance of E. coli by season and livestock type for more than one antibiotic, but regional differences were significant only for ampicillin. Over the three regions, discriminant analysis using MAR profiles correctly classified 27% of swine, 49% of poultry, 56% of beef and 51% of dairy isolates. Conclusions: Regional variations in MAR combined with moderate discrimination success suggest that MAR profiles of E. coli may only be marginally successful in identifying sources of faecal pollution. Significance and Impact of Study: This study demonstrates the existence of regional and seasonal differences in MAR profiles as well as the limited ability of MAR profiles to discriminate among livestock sources.