The position of mobile genetic elements (MGE) within eukaryotic genomes is often highly variable and we have exploited this phenomenon to develop a novel approach to strain differentiation in Toxoplasma gondii. Two PCR based strategies were designed in which specific primers were used to amplify T. gondii MGE's revealing information on element size and positional variation. The first PCR strategy involved the use of a standard two primer PCR while the second strategy used a single specific primer in a step-up PCR protocol. This approach was applied to T. gondii reference strains which were either acute virulent or avirulent to mice. The use of a standard two primer PCR reaction revealed the presence of a virulence related marker in which all avirulent strains possessed an additional 688 bp band. The single primer PCR strategy demonstrated that all virulent strains had identical banding patterns suggesting invariance within this group of strains. However, all avirulent strains had different banding patterns indicating the presence of a number of individual lineages within this group. The applicability and sensitivity of MGE-PCR in epidemiological studies was demonstrated by direct amplification of T. gondii from sheep tissue samples. All sheep isolates, tested in this way, gave identical banding patterns suggesting the presence of an endemic Toxoplasma strain on this farm.