The tobacco budworm, Heliothis virescens (F.), is one of the target pests of genetically modified cotton expressing Cry1Ac insecticidal protein (Bt cotton) derived from Bacillus thuringiensis Berliner. This study was conducted to evaluate the susceptibility of field-collected populations of H. virescens to Cry1Ac to establish a baseline for use in an insect resistance management program for Bt cotton in Brazil. Insects were sampled from the main Brazilian cotton-growing regions (Bahia, Mato Grosso, Mato Grosso do Sul, and Goiás) during the cropping seasons of 2007/08 and 2008/09. Cry1Ac susceptibility was estimated by using diet incorporation bioassays. H. virescens was highly susceptible to Cry1Ac protein. The estimated LC₅₀ values varied from 0.18 to 0.66 µg of Cry1Ac/ml of diet among the 2007–2008 populations (≈3.7-fold variation). Similarly, the EC₅₀ values based on growth inhibition ranged from 0.0053 to 0.0161 µg of Cry1Ac/ml of diet for the 2007–2008 populations («3.0-fold variation ). A joint analysis of the mortality data across all tested populations was used to develop and validate the diagnostic concentrations of 3.1 and 5.6 µg of Cry1Ac/ml of diet, the upper bound of the confidence interval and twice the LC₉₉ were selected, for resistance monitoring programs of H. virescens to Cry1Ac protein in Brazil.