A three year (2003–2005) field study compared the susceptibility to the Cry1Ab toxin, expressed in Bt maize, of Mediterranean corn borer (MCB) Sesamia nonagrioides populations collected from areas with different adoption rates of Bt maize in Spain with Bt-free areas in Greece. Spain is the only European country where the cultivar Compa CB derived from the event Bt176 was commercially grown, from 1998 to 2005. The large decrease of the titer of the toxin in this cultivar at later growth stages represented the worst-case scenario for resistance development of MCB, since larvae of the second and third generations were exposed to sublethal concentrations of Cry1Ab toxin. Our data revealed that the variation in susceptibility to Cry1Ab for the MCB Spanish field populations analyzed in the three years was very low, with LC50 values fluctuating between 12 and 30 ng Cry1Ab/cm2, regardless of the region of origin, the type of maize (Bt or non-Bt) and the year. Furthermore, no significant differences were found when comparisons were made with a laboratory population (LC50 values: 18–26 ng Cry1Ab/cm2) or with field populations from Greece (Bt-free areas), which displayed LC50 values ranging between 22 and 27 ng Cry1Ab/cm2. Standardizing bioassay protocols proved to be essential for obtaining comparable results. These findings suggest that resistant MCB populations did not evolve in those Spanish maize areas where Compa CB was largely cultivated for eight years, contradicting the expected rapid development of resistance under these unfavourable conditions. Additionally, our results can be used as baseline indices in post-market resistance monitoring programs if Bt maize is introduced in Greece. Further studies should continue, since the insights gained from a resistance monitoring program may help to enhance the durability of Bt maize.