Comparison of the publicly available genomes of the virulent Salmonella enterica serovar Typhimurium (S. Typhimurium) strains SL1344, 14028s and D23580 to that of the virulence-attenuated isolate LT2 revealed the absence of a full sequence of bacteriophage ST64B in the latter. Four selected ST64B regions of unknown function (sb7–sb11, sb46, sb49–sb50 and sb54) were mapped by PCR in two strain collections: (i) 310 isolates of S. Typhimurium from human blood or stool samples, and from food, animal and environmental reservoirs; and (ii) 90 isolates belonging to other serovars. The region sb49–sb50 was found to be unique to S. Typhimurium and was strongly associated with strains isolated from blood samples (100 and 28.4 % of the blood and non-blood isolates, respectively). The region was cloned into LT2 and knocked out in SL1344, and these strains were compared to wild-type isogenic strains in in vitro assays used to predict virulence association. No difference in invasion of the Int407 human cell line was observed between the wild-type and mutated strains, but the isolate carrying the whole ST64B prophage was found to have a slightly better survival in blood. The study showed a high prevalence and a strong association between the prophage ST64B and isolates of S. Typhimurium collected from blood, and may indicate that such strains constitute a selected subpopulation within this serovar. Further studies are indicated to determine whether the slight increase in blood survival observed in the strain carrying ST64B genes is of paramount importance for systemic infections.