Two candidate diagnostic concentrations of the Cry1Ab and Cry1Ac toxins from Bacillus thuringiensis corresponding to the LC₉₉ and EC₉₉ (effective concentration that causes 99% growth inhibition) for European corn borer, Ostrinia nubilalis (Hübner), were determined based on previously obtained baseline data. Validation experiments using field-collected European corn borer populations from across North America showed that for Cry1Ab, a concentration corresponding to the upper limit of the 95% confidence interval of the LC₉₉, produced mortality >99% for all populations tested. However, for Cry1Ac, adjustments and further validation are probably necessary. Development of B. thuringiensis resistance monitoring programs that rely on diagnostic techniques are discussed.