The Mediterranean corn borer, Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae), is a major pest of maize in the Mediterranean area. Transgenic Bt maize expressing the Cry1Ab toxin from the bacterium Bacillus thuringiensis can effectively control this pest. The characterization of S. nonagrioides population structure, at a large geographical scale, would provide some insight in decision making for resistance management. The genetic relationships among nine populations from Spain, one from France, one from Italy, three from Greece, and one from Turkey were assessed using Random Amplyfied Polymorphic DNA (RAPD) markers. Populations from France and Spain formed a cluster independent from a cluster of populations collected in Italy, Turkey, and Greece in a unweighted pair-group method with arithmetic average dendrogram constructed from Nei’s genetic distances. Average genetic differentiation among samples was significant for all geographical groupings analyzed (F_ST = 0.160 ± 0.014 for Spanish populations; 0.133 ± 0.022 for Spanish and French populations; and 0.095 ± 0.010 for Greek, Italian, and Turkish populations). Genetic differentiation was also significant for all paired comparisons of populations, including two Spanish populations separated by only 15 km with no apparent geographical barriers. No pattern of isolation by distance was observed among Mediterranean corn borer populations collected in Spain and France. These results suggest a limited genetic exchange between relatively distant S. nonagrioides populations in Europe, which might contribute to decreased rate of spread of resistance alleles once resistance has developed at a certain site.