Resistance of western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), to the Cry3Bb1 toxin derived from the bacterium Bacillus thuringiensis (Bt) that is produced by transgenic corn (Zea mays L), has been appearing in fields in Iowa, USA since 2009. As part of its insect resistance management plan for transgenic corn for the management of western corn rootworm, the Environmental Protection Agency requires resistance monitoring programs that must include the assessment of resistance using bioassays. In addition, the current understanding of resistance phenomena in western corn rootworm is based largely on computer modeling, interactions of susceptible insects with Bt corn, and experiments with resistant strains developed through laboratory based selection. We examined the discriminatory power of five bioassay methods, three single-plant methods and two seedling-mat methods, for detecting statistically significant differences in the magnitude of resistance in a Cry3Bb1 resistant, non-diapausing strain of western corn rootworm with field-evolved resistance (Hopkinton), compared to a non-diapausing, susceptible strain (Standard). In general, seedling-mat bioassays tended to better distinguish between Hopkinton and Standard on Bt corn than single-plant assays. Using the bioassay identified as being the most sensitive we measured the magnitude and inheritance of resistance of Hopkinton as well as second non-diapausing, Cry3Bb1 resistant strain of western corn rootworm with field-evolved resistance (Cresco). Additional diet-based bioassays were also used to measure the magnitude of resistance, and growth chamber experiments were used to measure several life history characteristics of western corn rootworm in the absence of Bt for the identification of fitness costs of Bt resistance. Hopkinton exhibited significantly greater resistance to Cry3Bb1 than Cresco. Non-recessive inheritance (h) was detected in Hopkinton, but not in Cresco. Additionally, fitness costs affecting development rate, survival to adulthood, and fecundity were identified in Cresco in the absence of Bt, whereas none were identified in Hopkinton. Non-recessive inheritance and few fitness costs will facilitate the rapid evolution of Cry3Bb1 resistance in the western corn rootworm. Data from these studies will aid in the development and assessment of insect resistance monitoring and management policies for western corn rootworm.