Genetic relatedness of enterococci from poultry litter to enterococci from nearby surface water and groundwater in the Lower Fraser Valley regions of British Columbia, Canada was determined. A new automated BOX-PCR and Pulsed-Field Gel Electrophoresis (PFGE) were used to subtype enterococcal isolates from broiler and layer litter and surface and groundwater. All surface water samples (n = 12) were positive for enterococci, as were 11% (3/28) of groundwater samples. Enterococcus faecium (n = 90) was isolated from all sources, while Enterococcus faecalis (n = 59) was isolated from all sources except layer litter. The majority of E. faecalis originated from broiler litter (28/59; 47.5%) while the majority of E. faecium were isolated from layer litter (29/90; 32.2%). E. faecalis grouped primarily by source using BOX-PCR. Isolates from water samples were dispersed more frequently among PFGE groups containing isolates from poultry litter. E. faecium strains were genetically diverse as overall clustering was independent of source by both molecular methods. Subgroups of E. faecium isolates based upon source (layer litter) were present in BOX-PCR groups. Three individual E. faecalis groups and two individual E. faecium groups were 100% similar using BOX-PCR; only one instance of 100% similarity among isolates using PFGE was observed. Although enterococci from litter and water sources were grouped together using BOX-PCR and PFGE, isolates originating from water could not be definitively identified as originating from poultry litter. Automation of BOX-PCR amplicon separation and visualization increased the reproducibility and standardization of subtyping using this procedure.