Cancer reflects the progressive accumulation of genetic alterations and subsequent genetic instability of cells. Differentiated thyroid cancers exhibit aneuploidy; cytogenetic studies have demonstrated the importance of genetic instability in thyroid cancer development. Pituitary tumour transforming gene (PTTG), also known as securin, is a mitotic checkpoint protein which inhibits sister chromatid separation during mitosis. PTTG is highly expressed in many cancers and over-expression of PTTG induces aneuploidy in vitro. Inter-(simple sequence repeat)-PCR (ISSR-PCR) allows simple and rapid screening of the human genome for genetic alterations. Using fluorescent-ISSR (FISSR) & ABI377 genotyping platform we investigated the relationship between the degree of genetic instability and PTTG expression in normal and tumour thyroid samples from 26 patients. The genomic instability index (GI-index) was 6.7-72.7% higher in cancers than normal, and in metastatic nodes from 2 patients were 104 & 225% greater than in the corresponding primary cancer. We also found that follicular thyroid tumours exhibited greater genetic instability than papillary tumours (27.6% versus 14.5%, p=0.03). By measuring PTTG mRNA using quantitative-PCR we demonstrated a relationship between PTTG expression and the degree of genetic instability in thyroid cancers (R2=0.80, p=0.007). To further investigate PTTG's role in genetic instability we transfected FTC133 thyroid follicular cells and observed increased genetic instability in cells over-expressing PTTG compared with vector-only controls (n=3, GI-Index VO= 29.7+/-5.2 versus WT-PTTG=63.7+/-6.4, p=0.013). Apoptosis assays demonstrated increased PTTG expression did not induce significant apoptosis. Overall, we describe the first use of FISSR-PCR in human cancers, and demonstrate increased genetic instability in differentiated thyroid cancers and metatstatic nodes compared with normal thyroid tissue. The degree of instability strongly correlated with PTTG-expression in these tissues. Furthermore, in vitro studies demonstrated up-regulation of PTTG in thyroid cells drives genetic instability. We conclude that PTTG may be an important gene in the mutator phenotype development in thyroid cancer.