Abstract: The olive fruit fly, Bactrocera oleae, is a major pest of olive crops and its expansion isrestricted to the geographic areas where olive trees are grown. The knowledge of the within andbetween populations genetic variability can help to understand the history of a species andmonitoring the origin and spread of invading populations. Such information could be crucial todefine appropriate strategies for eradication or control.In the present work, PCR amplification ofinter-simple sequence repeats (ISSR technique) was applied to the analysis of the geneticvariability of four Iberian populations of B. oleae. Flies from four different geographical areas,representing the Iberian distribution range of the species, were collected by harvesting infestedfruit and allowing the larvae to pupate in the laboratory. Four random primers (817, 820, 820,847, UBC primer set no. 9) were used to assess their genetic variation. The results showconsiderable levels of genetic polymorphism in the analysed samples, ranging from 78.5% to82.1%. Regarding the distribution of this variability, most of the genetic variation was foundwithin populations (92.6%). Likewise, a substantial level of gene flow (5.36) was deduced. Thus,ISSR are highly sensitive markers for variability detection in B. oleae and could help to answerfundamental questions related to the population structure and dynamics of the fly and, hence, toimprove management control.