Baseline susceptibility to the Cry1F toxin from Bacillus thuringiensis was determined for 11 European Union (EU) and 24 United States (US) populations of European corn borer, Ostrinia nubilalis (Hübner). The response to Cry1F exposure was measured as mortality and growth inhibition for each population. The aim of this study was to characterize inter-population variation in the susceptibility of EU and US European corn borer (O. nubilalis) neonates to Cry1F protein and additionally, intra-population variation for the EU European corn borer to help develop effective and sensitive resistance monitoring methods. In this study, neonate European corn borer were exposed to a range of concentration of microbially-produced Cry1F protein applied to the surface of artificial diet. Measures of mortality and growth inhibition of larvae on surface-treated diet relative to untreated control diet were taken after 7 days. A comparison between European and American corn borer populations showed that the range of variation in Cry1F toxin susceptibility assessed by mortality and growth inhibition was similar. Differences in Cry1F susceptibility between European corn borer populations across continents and among the various populations within each geography were apparent although similar variation among replicates for the same population were also observed in two EU populations. It is likely that these small differences reflect natural variation in sensitivity among populations of European corn borer as well as methodological differences and are not influenced by geographic location. The importance of establishing baseline susceptibilities to resistance detection is discussed.