A major national outbreak of multiresistant Salmonella enterica serovar Typhimurium definitive phage type 104 (MR DT104) occurred in England and Wales in the summer of 2000. Isolates of MR DT104 were characterised by antimicrobial resistance type (R-type), pulsed-field gel electrophoresis (PFGE), plasmid profiling and fluorescent amplified fragment length polymorphism (fAFLP) analysis. Results of R-type, PFGE and fAFLP showed that summer 2000 outbreak-associated isolates were indistinguishable from most MR DT104 isolates collected in England and Wales during the 1980s and 1990s. However, outbreak-associated isolates all had an additional 2-MDa plasmid (PP D), and this distinct profile allowed outbreak cases to be distinguished from background MR DT104 infections, thereby facilitating the epidemiological investigation by improving the specificity of the case definition. The study demonstrated the highly clonal nature of MR DT104 and the importance of a hierarchical approach to molecular subtyping for outbreak investigations.