Susceptibility to the Cry1Ac toxin from Bacillus thuringiensis in tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), is usually measured by performing bioassays under laboratory conditions. Accurate comparison of Cry1Ac susceptibility among H. virescens samples conducted in different places is challenged by several important methodological aspects, especially if different insect artificial diets are used to perform bioassays. In this study, we compared Cry1Ac susceptibility of four different-origin H. virescens colonies when challenged with this toxin incorporated into four different insect artificial diets. Our data show that Cry1Ac susceptibility was lower in all the H. virescens colonies for one of the commercial diets (Bio-Serv). Bio-Serv diet was one of the least significantly consumed diets by larvae of the four different colonies, which indicates that insects encountered less Cry1Ac toxin due to lower consumption of diet. Larvae fed Bio-Serv diet also seemed to display slower Cry1Ac toxin activation compared with larvae fed any of the other three diets tested. In contrast, a wheat germ-soybean diet (ARS) was one of the most consumed diets by the four H. virescens colonies. The increased consumption of ARS diet probably led to the high level of Cry1Ac susceptibility observed in all the H. virescens colonies. Our data highlight the importance of using common diets and use a standard tobacco budworm colony when comparing Cry1Ac susceptibility between diverse H. virescens strains or across time.