Technical Abstract: Fifteen microsatellite loci were used to genotype 108 accessions of cultivated olive, Olea europaea L. ssp. europaea var. europaea, and eight of O. europaea L. ssp. cuspidata (Wall. ex G. Don) Ciferri from the germplasm collection of the United States Department of Agriculture in Davis, California. Number of alleles per locus ranged from 3, for locus IAS-pOe12_A, to 16, for locus ssrOeUA-DCA11, and with an overall mean of 9.93. Observed heterozygosity ranged from 0.175, for locusUDO99–019, to 0.937, for locus GAPU89, with a mean of 0.640. The cluster analysis using the Unweighted Pair Group Method using Arithmetic mean (UPGMA) method displayed thirteen clusters within seven main groups that can be partially described by common geographic origin or fruit use though overlap among these groups was common. The locus-wise total gene diversity (HT) ranged from 0.319, at UDO99–019, to 0.847, at ssrOeUA-DCA3, with an overall mean of 0.696. Most of the gene diversity was partitioned within clusters, with proportions (HS/HT) ranging from 0.633, at IASpOe12_ B, to 0.848 at GAPU89 per locus, with a mean of 0.759. The principal components analysis explained 24.8% of the total variation along the first two components. Projection of accessions onto the first two principal components produced affinities generally in agreement with the results of the UPGMA cluster analysis. The California cultivar ‘Mission’ clustered closely with Iberian cultivars and may represent clonal selections adapted to local growing conditions. The results show significant diversity but low levels of differentiation among olive cultivars within the collection.