Eye colour is a highly transmissible and discernible trait in humans. A genome-wide association scan for variants associated to eye pigmentation was carried out on a large group of individuals coming from the Silk Road. Significant associations were detected not only with HERC2 (P-value=4.99 × 10(-37)) and OCA2 (P-value=4.51 × 10(-9)) genes but also with CTNNA2 gene (P-value=4.06 × 10(-8)). Moreover, the multifactor dimensionality reduction analysis clearly showed the effect of HERC2 haplotype over OCA2 mostly associated with SNP, thus enabling a highly accurate eye-colour prediction. Finally, the regression tree analysis showed that individuals carrying a given combination of haplotypes have a significant probability to show a blue or green/grey iris colour as compared with brown, with a gradient from west to east.