During 2009, Salmonella enterica subspecies enterica serovar Stanley isolates were recovered from cattle diagnostic specimens in southern Japan, and the isolates were examined to characterize the genetic determinants involved in this new pathogenicity that associated with mortality in cattle. All the isolates were multi-drug resistance exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, oxytetracycline, and kanamycin (ACSSuT-Km) encoded by blaTEM, catA, aadA1, sul1, tet(A), and aphA1 genes, respectively. Class 1 integrons of 1.5-kb size were detected in all MDR isolates. The isolates harboured easily transferable plasmids of ca. 210-kb with the potential of transmitting resistance phenotype and genotype detected in the donor isolates. XbaI-digested PFGE patterns generated two related clusters implicated in the dissemination of multi-drug resistance amongst Salmonella Stanley isolates. An emergence of multi-drug resistant Salmonella Stanley amongst food-producing animals, including cattle is a threat to human health, as resistant isolates may be transmitted to humans through the food chain.