The tephritid Bactrocera oleae (Gmelin) is a harmful pest of olive crops that cause important agricultural and economic losses in the Mediterranean area where 90% of the world olive trees are cultivated. The knowledge of the genetic diversity in insect pest species populations is critical for decisions concerning appropriate control management strategies. In the present work, the genetic variability within and among 7 populations -five from Spain, one from Italy and one from Tunisia-, was assessed by sequencing 1151 bp of the COI gene. A total of 21 haplotypes were observed. The intraspecific diversity was high, particularly in the Spanish populations (haplotype and nucleotide diversity 0.84 and 0.00137, respectively). However, the genetic differentiation among the populations was low in the case of Spanish ones (Fst between 0 and 0.041), and higher –and statistically significant– when comparing with the Italian and Tunisian samples. The haplotypes distribution and the PCoA analysis show three clear groups of populations: Spanish, Italian and Tunisian. The results might indicate the length of time elapsed since B. oleae became established in the Mediterranean region, the large effective sizes expected of its populations and the high gene flow among Iberian populations. The information could be relevant for integrated control programmes coordination.