An in vivo ALS assay was used to differentiate between susceptible and resistant kochia biotypes from Idaho and Montana. Experiments were also conducted using this assay to determine the effect of the timing of herbicide application on ALS activity in resistant and susceptible cocklebur biotypes from Mississippi. When treated with chlorsulfuron, resistant kochia biotypes demonstrated 160-and 170-fold resistance at the whole plant level for the Idaho and Montana biotypes, respectively, compared to susceptible biotypes. Using the in vivo ALS assay, the Idaho and Montana biotypes showed 70- and 490-fold resistance, respectively, based on I50 values. The biotypes also demonstrated a low level of cross-resistance to imazethapyr using the in vivo technique. Resistant cocklebur biotypes showed approximately 200-fold resistance to imazaquin compared with susceptible biotypes. This research demonstrated that the in vivo assay can be used effectively to identify resistant biotypes 6 and 24 h after treatment, and to determine the extent of cross-resistance.