Multiresistant Salmonella enterica subspecies enterica serovar Typhimurium definitive type 104 (S. Typhimurium DT104 or DT104) bacteria are important pathogens in animals and humans. DT104 isolates are often called pentaresistant strains that spread clonally. The purpose of this study was to determine phenotypic, genotypic, and epidemiologic characteristics of 175 S. Typhimurium DT104 strains isolated from food-producing animals in Canada. More than 90% of the isolates were resistant to ampicillin (Amp), chloramphenicol (Chl), florfenicol (Flo), sulfisoxazole (Sul), and tetracycline (Tet), 53% of the isolates were additionally resistant to spectinomycin (Spc) and streptomycin (Str), and 28% to kanamycin (Kan) and neomycin (Neo). Sixty-one percent of the strains harbored a single 60-MDa plasmid, 21% contained 60- and 2.0-MDa plasmids, and 4% had 60, 4.6- and 2.0-MDa plasmids. Resistance to Kan and Neo was encoded by the aminoglycoside aphA-1 gene on 2.0-MDa plasmids, whereas resistance to trimethoprim (Tmp) and Sul was encoded by the dhfrIb gene on 4.6-MDa plasmids. Polymerase chain reactions (PCR) showed the presence of integrons with the ant (3")-Ia aminoglycoside adenyltransferase and the bla(PSE-1) beta-lactamase gene cassettes, and the presence of the flost gene in all but one strain resistant to Spc and Str, Amp, and Chl and Flo, respectively. DT104 isolates from cattle at six feedlots represented a separate clone; they were sensitive to Str and Spc and lacked the ant (3")-Ia gene. Pulsed-field gel electrophoresis (PFGE) using Bln I, Spe I, and Xba I resulted in 15, 12, and 8 PFGE patterns, respectively. In summary, we observed considerable diversity in phenotypic, genotypic, and epidemiological characteristics among the DT104 isolates.