Seven polymorphic microsatellites were developed in olive. Six of them came from a genomic library enriched for GA and CA repeat sequences. They showed single locus polymorphism in a set of 23 olive cultivars (from six to nine alleles per locus). Three different pairs of loci were sufficient to discriminate all cultivars. The other polymorphic primer pair was designed from a published sequence for olive lupeol sgutase and revealed just two alleles. The seven primer pairs were tested on two accessions of five other species of the Oleaceae and three, EMO2, EMO13 and EMO90, revealed polymorphism in two, four and three species, respectively.