The sugarcane borer, Diatraea saccharalis (F.), is a key pest of sugarcane and other grasses, which is a current focus of research aiming to develop transgenically resistant genotypes. However, the current susceptibility of Brazilian populations of the sugarcane borer to Bt toxins is unknown. Laboratory assays were carried out to characterize the susceptibility of sugarcane borer to the Bt toxin Cry1Ab and to select a resistant strain for additional studies on Cry1Ab resistance. Susceptibility was characterized by exposing neonates of different Brazilian colonies of sugarcane borer to different concentrations in meridic diet using surface application of Cry1Ab toxin. Selection for Cry1Ab resistance was carried out by exposing neonates to Cry1Ab-expressing maize (MON 810). The resistance of the borer populations to Cry1Ab was variable with LC50 and EC50 values reaching about 30-fold. The larvae responded positively to Cry1Ab selection exhibiting a 55-fold increase in resistance after four generations. This suggests the suitability of using leaves containing Bt-expressing genes for selection and the existence of variability of Bt-resistance in populations of the sugarcane borer.