Inter–Simple Sequence Repeat (ISSR) molecular markers and morphological analysis were used in order to characterize wild populations and cultivated forms of orphan crop species Capparis spinosa L. in a Mediterranean island complex. Nineteen wild populations belonging to two different subspecies, C. spinosa subsp. spinosa and subsp. rupestris, were sampled in different environments in Sicily and the surrounding islets Lampedusa, Pantelleria and Salina. Different biotypes cultivated in Pantelleria and Salina were analyzed. Six ISSR primers were selected for genetic characterization, and all clear and reproducible bands were scored and analyzed. Among the 47 ISSR bands obtained, 97.5% were polymorphic. Results of AMOVA and STRUCTURE analysis suggested a clear genetic distinctness between subspecies at the regional level and suggested the existence of two taxonomic groups among wild populations, with different ecological preferences and distinctive morphological characters. Cultivated forms showed genetic affinity to subsp. rupestris. ISSR analysis not only provided specific molecular markers to discriminate the taxa, but also proved useful in supporting the hypothesis of a hybrid origin of the intermediate phenotypes found in overlapping distribution areas. The identified molecular markers provided a basic tool for the DNA fingerprinting of wild and commercial capers in the Mediterranean region and nearby territory.