Type 2 diabetes (T2D) has been the subject of numerous genetic studies in recent years which revealed associations of the disease with a large number of susceptibility loci. We hereby initiate the evaluation of T2D susceptibility loci in the Greek-Cypriot population by performing a replication case-control study. One thousand and eighteen individuals (528 T2D patients, 490 controls) were genotyped at 21 T2D susceptibility loci, using the allelic discrimination method. Statistically significant associations of T2D with five of the tested single nucleotide polymorphisms (SNPs) ( rs7901695, rs8050136, rs5015480, rs13266634 and rs4402960) were observed in this study population. Furthermore, 14 of the tested SNPs had odds ratios (ORs) in the same direction as the previously published studies, suggesting that these variants can potentially be used in the Greek-Cypriot population for predictive testing of T2D. In conclusion, our findings expand the genetic assessment of T2D susceptibility loci and reconfirm five of the worldwide established loci in a distinct, relatively small, newly investigated population.