Several experiments were conducted to evaluate the utility of an in vivo acetolactate synthase (ALS) assay for comparing sensitivity to imazamox among imidazolinone-resistant wheat cultivars/lines. Ten single-gene imidazolinone-resistant winter wheat cultivars/lines, one two-gene and four single-gene imidazolinone-resistant spring wheat cultivars/lines, and three pairs of heterozygous and homozygous imidazolinone-resistant winter wheat lines were evaluated in the assay experiments. Additionally, a dose-response assay was conducted to evaluate the tolerance of several imidazolinone-resistant wheat cultivars to imazamox on a whole plant level. The I50 value (i.e., the imazamox dose that inhibited ALS activity by 50%) of the winter wheat cultivar ‘Above’ was 54 to 84% higher than the I50 values of 99-420, 99-433, and CV-9804. However, based on the results of this study, it is unclear whether genetic background or market class (hard red winter vs. soft white winter) influences the level of ALS inhibition by imazamox. Teal 15A, the two-gene imidazolinone-resistant spring wheat cultivar, had an I50 value that was two to three times greater than the I50 value of the single-gene imidazolinone-resistant spring wheat cultivars/lines. The heterozygous imidazolinone-resistant wheat lines had I50 values that were 69 to 81% less than the I50 values of the homozygous lines. In the whole plant dose response, the R50 values (i.e., the imazamox dose that reduced biomass by 50%) of the susceptible cultivars Brundage 96 and Conan were 15 to 17 times less than the homozygous single-gene imidazolinone-resistant winter and spring cultivars/lines, whose R50 values were about 1.7 times less than the R50 value of the two-gene imidazolinone-resistant spring wheat line, Teal 15A. The results of the in vivo ALS imazamox assays and the whole plant imazamox dose-response assay were similar, indicating that the in vivo assay can be used to accurately and quickly compare resistance between imidazolinone-resistant wheat cultivars/lines.