Germplasm collections are basic tools for conservation, characterization, and efficient use of olive genetic resources. The identification of the olive cultivars maintained in the collections is an important ongoing task which has been performed by both, morphological and molecular markers. In the present study, based on the sequencing results of previous genomic projects, a new set of 1,043 EST-SNP markers has been identified. In order to evaluate its discrimination capacity and utility in diversity studies, this set of markers was used in a representative number of accessions from 20 different olive growing countries and maintained at the World Olive Germplasm Collection of IFAPA Centre 'Alameda del Obispo' (Córdoba, Spain), one of the world's largest olive germplasm bank. Thus, the cultivated material included: cultivars belonging to previously defined core collections by means of SSR markers and agronomical traits, well known homonymy cases, possible redundancies previously identified in the collection, and recently introduced accessions. Marker stability was tested in repeated analyses of a selected number of accessions, as well as in different trees and accessions belonging to the same cultivar. In addition, 15 genotypes from a cross 'Picual' × 'Arbequina' cultivars from the IFAPA olive breeding program and a set of 89 wild genotypes were also included in the study. Our results indicate that, despite their relatively wide variability, the new set of EST-SNPs displayed lower levels of genetic diversity than SSRs in the set of olive core collections tested. However, the EST-SNP markers displayed consistent and reliable results from different plant material sources and plant propagation events. The EST-SNPs revealed a clear cut off between inter- and intra-cultivar variation in olive. Besides, they were able to reliably discriminate among different accessions, to detect possible homonymy cases as well as efficiently ascertain the presence of redundant germplasm in the collection. Additionally, these markers were highly transferable to the wild genotypes. These results, together with the low genotyping error rates and the easy and fully automated procedure used to get the genotyping data, validate the new set of EST-SNPs as possible markers of choice for olive cultivar identification.