Forty olive (Olea europaea L.) cultivars from Valencia, Spain, were screened using random amplified-polymorphic DNA (RAPD) markers. Eighteen selected decamer primers produced 34 reproducible amplification fragments that were then used as polymorphic markers. The resulting combinations of these RAPD markers were used to discriminate 40 cultivars. Results were analyzed for similarity among cultivars and the relatedness of polymorphisms obtained between cultivars agreed with previous results using isozymes. Unweighted pair group method cluster analysis of their similarity values revealed two main groups divided according to geographic origin within Valencia. A third group, which included two Spanish cultivars from regions outside of Valencia, was clustered separately from the Valencian cultivars. RAPD technology proved useful in discriminating closely related cultivars. There was no apparent clustering of cultivars by fruit size or other morphological traits.