Salmonella enterica ser. Typhimurium was isolated from freshwater sediments and chromosomally labelled with a stable variant of the green fluorescent protein (GFP). The pUT mini-Tn5 Km transposon was used to introduce the gfp gene onto the chromosome of the S. Typhimurium strain by tri-parental mating. Southern Blot hybridisation confirmed that the gene had integrated into the chromosome. The gfp gene was stably maintained and the labelled strain was not growth-rate impaired. The incorporation of the gfp gene did not convey any significant loss of phenotype which would affect the survival and behaviour of the tagged strains. The tagged S. Typhimurium strain was used to spike an established drinking water biofilm and was able to colonise and persist within the biofilm. The tagged strain was also successfully used to study the survival of S. Typhimurium in natural sediments under different temperatures. These tagged strains can therefore be used to study the fate and survival of different Salmonella strains in water environments.