Brown and blue eye colours are primarily explained by the single nucleotide polymorphism (SNP) HERC2 rs12913832. However, the genetics of eye colours that appear to be neither blue nor brown are not well understood. In this study, 230 unrelated Italian individuals were typed for 32 SNP loci in pigmentary genes. High resolution digital images of the participants’ eyes were taken and the iris region was successfully extracted with the use of the custom designed software Digital Iris Analysis Tool (DIAT) from 218 of the 230 (95%) images. The software counted the numbers of blue and brown pixels in the iris region and calculated a Pixel Index of the Eye (PIE-score) that described the eye colours quantitatively. The PIE-score ranged from _1 to 1 (brown to blue). We investigated the association of the PIE-scores extracted from the eye images with the genotypes of the 32 pigmentary SNPs. We observed a statistically significant association between the PIE-scores and the SNP loci rs12913832, rs4778241, rs7495174 in the HERC2/OCA2 region and the locus rs16891982 in SLC45A2.