Majority of Australian Salmonella Typhimurium (S. Typhimurium) outbreaks are linked with the consumption of contaminated eggs or egg products. Multi Locus Variable Number Tandem Repeat Analysis (MLVA) has been the preferred method for trace back investigation of outbreaks. The aims of this study were to determine if in vivo passage of S. Typhimurium in laying hens led to changes in MLVA pattern and whether there were subsequent changes in virulence. Hens were infected at 14 weeks post hatch with either 109 colony forming units (CFU) of S. Typhimurium or 109 CFU of an equal suspension of both S. Typhimurium and S. Mbandaka. Faecal samples were collected from each bird at days 1, 3, 6, 9, 12 and weeks 3, 5, 7, 9, 11, 13 and 15 post-infection (p.i.). Faecal shedding of S. Typhimurium was highest in both treatment groups at day 6 p.i. and decreased from there on. Shedding was significantly higher in the single infection group (P ≤ 0.01). MLVA typing of S. Typhimurium isolates collected during the trial revealed a change in MLVA pattern at week 9 p.i. onwards in bacteria isolated from the co-infection treatment group (from 03-24-11-11-523 to 03-24-12-11-523). Human intestinal epithelial cells (Caco2) were used for invasion assays, with this assay being designed to characterise potential changes in invasion, one aspect of bacterial virulence. No statistically significant difference in invasion was observed between different MLVA types, however percent invasion was variable across all isolates. The change in MLVA pattern occurred after only one in-vivo co-infection passage of the isolate thus, the co-infection environment may be responsible for driving MLVA change.