Salmonella enterica serovar Typhimurium phage type DT41 has previously been identified from salmonella-positive broiler breeder flocks in Denmark and isolates obtained from different flocks have demonstrated major diversity by multiple-locus variable-number tandem-repeats analysis (MLVA) typing. To elucidate whether the high diversity observed by MLVA was related to multiple independent introductions at farm level or genetic instability of markers, we investigated the genomic stability of different clones of S. Typhimurium DT41. In the in vitro genomic stability experiment, feed pellet- and dust samples inoculated with four strains of DT41 were kept at three different temperatures. The in vitro genomic stability was also assessed by conducting a serial passage experiment. In a subsequent in vivo experiment, broiler breeders of three different age groups were challenged with a strain of poultry and human origin, respectively. The in vitro experiment demonstrated that DT41 survived more than 6 months in feed-pellets at 20 °C whereas the survival in dust was less than 4 weeks. Infection pattern and excretion varied for the poultry and human strain and birds of different age groups as revealed by the in vivo experiment. Genetic stability of cultures obtained from the in vitro and in vivo survival/passage was investigated by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and MLVA. The results of plasmid profiling and PFGE demonstrated genomic stability of all but one strain kept in dust at 20 °C for 3 weeks. Minor genetic changes were observed in isolates from the in vitro experiment as revealed by MLVA. The epidemiological impact of these findings is briefly discussed.