Salmonella enterica serovar Gallinarum causes a severe systemic disease, fowl typhoid, primarily in chickens and turkeys, and it remains a disease of worldwide significance. Multilocus variable-number tandem-repeat analysis (MLVA) has proved to be very useful for subtyping other Salmonella serovars. We describe the development of a simple MLVA assay for S. enterica serovar Gallinarum that is comparable with pulsed-field gel electrophoresis (PFGE) in resolution. The genome sequence of S. enterica serovar Gallinarum strain 287/91 was analysed for potential variable-number tandem repeats (VNTRs) and then polymerase chain reaction assays were developed to assess the variability of the loci. Four VNTR markers were selected and used in a multiplex fragment analysis assay. The stability of the VNTR markers was assessed by conducting in vitro passage experiments with two strains (95 clones per strain) over a 30-day period. A MLVA of 68 strains of S. enterica serovar Gallinarum based on the four VNTR loci distinguished 26 allelic profiles. The MLVA assay showed a Simpson's diversity index of 0.918, whereas PFGE analysis produced 23 patterns and had a diversity index of 0.874. Most importantly, the MLVA further discriminated strains having the same PFGE pattern. The MLVA assay is a highly discriminatory genotyping method for S. enterica serovar Gallinarum. Therefore, MLVA can be a useful addition to routine PFGE analysis for molecular epidemiological investigation of fowl typhoid.