Two new insect colonies were created by separating virgin western corn rootworm, Diabrotica virgifera virgifera LeConte, males and females from both a selected laboratory colony that was being reared on eCry3.1Ab-expressing corn (Zea mays L.) and a control colony reared on its near-isoline corn. Females from the selected colony were paired with males of the control colony and vice versa to create both a selected female by control male colony (Sel♀) and control female by selected male colony (Con♀). Both colonies along with their parental colonies (eCry3.1Ab-selected and control) were evaluated on eCry3.1Ab-expressing corn and its near-isoline in seedling assays. Larvae from each colony were also used in diet toxicity experiments in order to determine the LC₅₀ and EC₅₀ values for the eCry3.1Ab toxin for each. Statistical analysis of seedling assay experiments did not indicate any significant colony × corn interaction but did show a significant main effect of corn type for both larval recovery and larval head capsule widths. Results from the diet toxicity assays showed the control colony to have a significantly lower LC₅₀ value than the selected and cross colonies and a significantly lower EC₅₀ than the selected and Con♀ colonies. Calculations of dominance values (h) of eCry3.1Ab resistance traits from seedling assays indicated that the two reciprocal cross colonies have a dominance value (h) of ∼1, suggesting dominance of the eCry3.1Ab resistance trait.