In summer 2004, an outbreak caused by Salmonella enterica serovar Enteritidis phage type 13 (S. Enteritidis PT13) was recorded in the Czech Republic. As well being a relatively rare phage type the strain was also ampicillin resistant. Outbreak (n = 39) and pre-outbreak isolates (n = 13) were characterized by pulsed-field gel electrophoresis (PFGE), β-lactamase gene polymerase chain reaction and plasmid profile. The majority of outbreak isolates (n = 37) were identical in XbaI PFGE profile, and two other outbreak isolates each differed from this profile by one or two fragments respectively. The pre-outbreak isolates were uniform in PFGE profile but distinct from the outbreak strain. Ampicillin resistance was confirmed to be encoded by the blaTEM gene located on the TnA transposon. This gene was readily transferable to a S. Enteritidis recipient strain and was associated with the transfer of a 200-kb plasmid. Our results indicate that all S. Enteritidis PT13 tested from 2004 belonged to a single outbreak strain which prior to 2004 had not been recognized in the Czech Republic.