SummaryTo assess genetic relatedness in thornless blackberry (Rubus spp.), 11 different blackberry cultivars were screened using random amplified polymorphic DNA (RAPD) markers. The blackberries selected represented four different thornless backgrounds. Genetic similarity was estimated using 140 random primers, and the cluster analysis conducted using the RAPD data grouped the cultivars into three distinct clades. Ninety-eight primers produced 113 cultivar-specific RAPD fragments capable of identifying each cultivar. In addition, reproducible polymorphism using two primers was observed within the ‘Evergreen’ (R. laciniatus) clade that consisted of the pure thornless blackberry ‘Everthornless’, the chimeral ‘Thornless Evergreen’, and their thorny progenitor ‘Evergreen’. All three plants are believed to be identical, except for a single mutational event that caused the phenotypic change from thorny to thornless. The R. laciniatus RAPD marker data provide information that may eventually be useful to identify the gene(s) responsible for thornlessness in that species.