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Abstract: From 2005 to 2015, Ostrinia nubilalis were collected in the most important maize‐growing areas in Europe where MON 810 was cultivated. The susceptibility of these O. nubilalis collections to the Cry1Ab protein was determined using overlay bioassays and compared to that of reference (control) strains. Larvae that died or did not moult after 7 days were used to calculate a moulting inhibition concentration (MIC). Two different batches of Cry1Ab protein were used over the course of this study. Between 2005 and 2015, 145 collections of O. nubilalis from 14 areas were analysed. The Cry1Ab susceptibility of populations from different geographic regions differed only slightly across years. The greatest variability in the MIC50 for field samples collected from 2005 to 2011 and tested with batch 1 was 6.6‐fold in 2006. For field‐collected O. nubilalis, the difference between MIC50 values of the most susceptible and most tolerant samples was 13.1‐fold for this period. For samples collected in 2012–2015 and tested with batch 2, the greatest variability was 4.1‐fold in 2014. A diagnostic concentration (MIC99) was calculated for batch 1 (48 ng/cm2) using the results from all the collections in 2005–2012. Bridging experiments indicated that the diagnostic concentration for batch 2 was 28 ng/cm2. From 2006 onwards, no O. nubilalis reached the 2nd larval stage when the diagnostic concentration of either batch of Cry1Ab was used. Only one insect collected from Romania in 2012 and two insects collected as reference strain from Spain in 2015 survived exposure to a dosage of 20 ng/cm2, and none of these larvae survived on MON 810 maize. Our results indicate that there has been no significant change in susceptibility to Cry1Ab in European populations of O. nubilalis over the period 2005–2015. [ABSTRACT FROM AUTHOR]