The genetic diversity existing in the cultivated olive (Olea europaea L.) is enormous. For some time now, scientists have made use of DNA-based markers for a better genetic knowledge of olive. The goal of this report is to announce 12 new SSRs in olive and evaluate their polymorphism within the species. A genomic library was enriched for GA, GT and ACT repeats sequences. Primer pairs were designed for 24 sequences containing SSR. Four of them failed to amplify, eight produced spurious bands or complex bands patterns and 12 succeeded in giving reliable PCR products. Considering these 12 primer pairs, ten of them showed single locus amplification whereas the other two revealed two loci each. The multi-locus nature of these two SSRs and their inheritance fashion were studied in progenies from 'Manzanilla de Sevilla' × 'Arbequina' and 'Picual' × 'Arbequina' crossings. All the expected classes were found in the segregation of the four loci evaluated. Only one locus did not exhibit Mendelian inheritance and the presence of a null allele was confirmed in the paternal genotype of the second cross. A total of 85 alleles were detected for these 12 SSRs when analyzing 51 olive cultivars. The number of alleles per locus ranged from 1 to 15. Twenty-five out of the 85 alleles identified were unique. The expected heterozygosity varied between 0 and 0.835.