Abstract The tephritid Bactrocera oleae (Gmelin) is a harmful pest of olive crops; its larvae are monophagous and feed exclusively on olive fruits. Despite the economic importance of this species, important issues remain to be clarified. In the present study, the genetic variability within and among 21 populations was assessed using random amplified polymorphic DNA–PCR (seven primers). A considerable level of intraspecific diversity was detected (at population level: P = 51–70%, Hpop = 4.61; at species level: P = 63%, Hsp = 5.38) but the genetic differentiation among the populations was low (Shannon’s diversity index 14%,amova4–8%). However, the dendrogram and principal components analysis reflect some interesting points. The most southerly of the Mediterranean populations (Tunisia) differ significantly from the remaining populations. The general results might be explained by the length of time that has elapsed as B. oleae became established in the Mediterranean region, the large effective sizes expected of its populations and gene flow among these populations. The results strongly suggest the existence of a single, large Northern Mediterranean olive fly population rather than several small, isolated populations and have a significant value in terms of control practices.