Salmonella enterica serotype typhimurium (S. typhimurium) DT104 (DT104) first emerged as a major pathogen in Europe and is characterized by its pentadrug-resistant pattern. It has also been associated with outbreaks in the United States. The organism typically carries resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. The mechanism of chloramphenicol resistance in DT104 Has determined by producing antibiotic-resistant Escherichia call host strain clones from DT104 DNA, DNA from chloramphenicol-resistant clones was sequenced, and probes specific for the genes flo(S. typhmurium) (flo(St)), int, invA, and spvC Here produced for colony blot hybridizations. One hundred nine Salmonella isolates, including 44 multidrug-resistant DT104 isolates, were tested to evaluate the specificities of the probes. The gene flo(St), reported in this study, confers chloramphenicol and florfenicol resistance on S. typhimurium DT104. Florfenicol resistance is unique to S. typhimurium DT104 and multidrug-resistant S. typhimurium isolates with the same drug resistance profile among ail isolates evaluated. Of 44 DT104 isolates tested, 98% were detected based on phenotypic florfenicol resistance and 100% had the flo(St)-positive genotype, Resistances to Borfenicol and chloramphenicol are conferred by the gene flo(St), described in this paper. Presumptive identification of S. typhimurium DT104 can be made rapidly based on the presence of the flo(St) gene or its resulting phenotype.