Molecular methodologies are increasingly being developed to assess the origin and authenticity of raw organic materials and processed food products. Here we describe the application of a microarray-based assay for single nucleotide polymorphisms (SNPs) identification in olive cultivars. The assay distinguishes alleles in a ligation detection reaction (LDR), with subsequent fluorescent detection by hybridization on a universal array (UA). The LDR-UA approach was used to detect 17 SNPs in olive genomic sequences previously amplified by PCR from fresh olive leaves. Genotype calls obtained with the LDR-UA were in full agreement with those determined by direct sequencing. The panel of 17 SNPs is sufficient to discriminate 49 olive varieties selected among the most widely cultivated for olive oil production in the Mediterranean area.