Eggplant (Solanum melongena Linn.), or brinjal, was engineered to express an insecticidal protein (Cry1Ac) from Bacillus thuringiensis (Bt) and commercialized in Bangladesh on a limited basis in 2014. As part of an insect resistance management strategy, studies were conducted to determine the susceptibility of the targeted insect pest, the eggplant fruit and shoot borer, Leucinodes orbonalis (Guenée), to Cry1Ac using a diet-incorporation bioassay method. Eighteen populations of L. orbonalis were collected from the main brinjal growing areas in 17 districts of Bangladesh during 2018–2019 and assayed. Larvae from each population were reared to adults and allowed to mate. Eggs from the matings were allowed to hatch, and neonates were used for bioassays. Bioassays were performed with different concentrations of Cry1Ac and an untreated control. Median lethal concentrations (LC50) ranged between 0.035 and 0.358 ppm and molt inhibitory concentration (MIC50) values ranged from 0.008 to 0.181 ppm. Variation in susceptibility among field populations was 10.22-fold for LC50 and 22.63-fold for MIC50. These results were compared to values from 73 populations in India. Overall, the results showed similar natural variation and suggest that these Bangladeshi values can be used as benchmarks for resistance monitoring as Bt brinjal becomes more widely adopted in Bangladesh.