In the present work, a set of eight new hexa-nucleotide simple sequence repeats (SSRs) is reported in olive (Olea europaea L). These SSRs loci were generated on the basis of expressed sequence tag (EST) sequences in the frame of an olive genomic project. The markers showed a high level of polymorphism when tested on a set of cultivars used as genitors in the olive breeding program of Córdoba, Spain. The long-core repeat motif of these markers allows a wider separation among alleles, thus permitting an accurate genotyping. Besides, these markers showed comparable levels of polymorphism to di-nucleotide SSRs, the only ones so far reported in olive. Selected on the basis of their discrimination capacity, four of the eight SSRs were used to test their ability for paternity testing in a total of 81 seedlings coming from 12 crosses. The paternity testing showed that seven crosses matched the alleged paternity and the remaining five were products of illicit pollinations. These results exactly matched with previous paternity testing performed with di-nucleotide SSR markers. These results demonstrate the usefulness of the developed hexa-nucleotide repeated motifs for checking the paternity of breeding progenies and suggest their use on variability studies.