Salmonella Typhimurium definitive type 104 (DT104) has emerged as a common cause of salmonellosis in humans and cattle, yet previous reports involving horses are sparse. This study reports the emergence of DT104 as an important pathogen in horses in Ontario. The first clinical case of DT104 infection at the Ontario Veterinary College was identified in 1997. Seventeen cases of DT104-associated salmonellosis were identified between 1997 and 2000. In 2000, 12 of 13 cases of salmonellosis were due to DT104. Salmonellosis in horses due to DT104 is of concern, since the organism is multiresistant to antibiotics and poses increased zoonotic risk. Phage type distribution of Salmonella isolates should be monitored to determine whether DT104 will remain a prevalent equine pathogen.