Phenotypic characterisation of germplasm collections is a decisive step towards association mapping analyses, but it is particularly expensive and tedious for woody perennial plant species. Characterisation could be more efficient if focused on a reasonably sized subset of accessions, or so-called core collection (CC), reflecting the geographic origin and variability of the germplasm. The questions that arise concern the sample size to use and genetic parameters that should be optimized in a core collection to make it suitable for association mapping. Here we investigated these questions in olive (Olea europaea L.), a perennial fruit species. By testing different sampling methods and sizes in a worldwide olive germplasm bank (OWGB Marrakech, Morocco) containing 502 unique genotypes characterized by nuclear and plastid loci, a two-step sampling method was proposed. The Shannon-Weaver diversity index was found to be the best criterion to be maximized in the first step using the CORE HUNTER program. A primary core collection of 50 entries (CC 50) was defined that captured more than 80% of the diversity. This latter was subsequently used as a kernel with the MSTRAT program to capture the remaining diversity. 200 core collections of 94 entries (CC 94) were thus built for flexibility in the choice of varieties to be studied. Most entries of both core collections (CC50 and CC 94) were revealed to be unrelated due to the low kinship coefficient, whereas a genetic structure spanning the eastern and western/central Mediterranean regions was noted. Linkage disequilibrium was observed in CC 94 which was mainly explained by a genetic structure effect as noted for OWGB Marrakech. Since they reflect the geographic origin and diversity of olive germplasm and are of reasonable size, both core collections will be of major interest to develop long-term association studies and thus enhance genomic selection in olive species.