A study was conducted to characterize 318 Salmonella enteritidis strains that were mainly isolated from poultry and their environment in Canada. Biotype, phagetype (PT), plasmid profile (PP), hybridization with a plasmid-derived virulence sequence probe, antibiotic resistance, outer membrane proteins (OMPs), and lipopolysaccharide (LPS) profiles were determined. Relationships of these properties to one another, and their diagnostic and pathogenic significance were assessed. Biotyping indicated that failure to ferment rhamnose was sometimes useful as a marker for epidemiologically related strains. Phagetyping was the most effective method for subdividing S. enteritidis; it distinguished 12 PTs. Phagetype 13 was occasionally associated with septicemia and mortality in chickens. The strains belonged to 15 PPs. A 36 megadalton (MDa) plasmid was found in 97% of the strains. Only the 36 MDa plasmid hybridized with the probe. Seventeen percent of the strains were drug resistant; all strains were sensitive to ciprofloxacin. Thirty-five of 36 strains possessed the same OMP profile, and 36 of 41 strains contained smooth LPS.