In wheat (Triticum aestivum L.), cultivars resistant to imidazolinone herbicides are an important tool for weed management. The target site of imidazolinones is acetohydroxyacid synthase (AHAS), an enzyme coded by three homeologous genes (ahasL‐A1, ahasL‐B1, and ahasL‐D1). This study aimed to evaluate the effect of the imidazolinone herbicide imazamox on a single‐gene (AhasL‐D1) resistant line (R1) and a two‐gene (AhasL‐B1 and AhasL‐D1) resistant line (R2) at multiple biological levels. These lines showed variation in the transcriptional levels of ahasL homeologs, but no differences in total AHAS activity. The relative contribution of resistant alleles to the ahasL transcript pool accounted for 47% in the R1 line and 77% in the R2 line. The in vitro AHAS inhibition of the R1 line was intermediate between the R2 and the susceptible lines. At an anatomical level, high imazamox concentrations induced alterations in the root tip features of R1 plants but did not affect R2 plants, whereas at in vivo AHAS activity and whole‐plant levels, both resistant lines showed high resistance. The herbicide dose that reduced shoot biomass of resistant lines by 50% was 35‐fold higher than that of the susceptible line. We conclude that homeolog variation allows a higher contribution of resistance genes to the ahasL transcript pool in the R1 and R2 lines. Although both resistant lines showed high whole‐plant resistance, the differential root damage induced by imazamox between wheat lines suggests that two‐gene resistant cultivars could be an effective option to avoid the negative impacts caused by herbicide residues in the soil.