The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is considered one of the most economically damaging pests of citrus orchards in Spain. The characterization of C. capitata population structure, at a large geographical scale, by using a combination of RAPD and ISSR markers can allow analyzing the genetic variability of this species, and provide some insight in decision making for resistance management, recently recorded in Spain. We compared six Spanish populations along the Mediterranean area (Gerona, Amposta, Tortosa, Castellón, Valencia and Málaga) with populations from other geographical areas where this pest is widely distributed (Africa, Middle East, South America and Atlantic Islands) and two laboratory strains. The results obtained with both types of molecular markers were similar. A dendrogram based on Nei genetic distances showed that all Mediterranean Spanish populations, except the population collected in Gerona, were clearly separated from the rest. However, no clear differentiation among Spanish populations was found, probably as a result of the high levels of gene flow (Nm value of 2.8 for RAPD and 3.9 for ISSR). Implications of these findings on resistance management of C. capitata are discussed.