The rapid evolution and spread of glyphosate-resistant (GR) kochia in the Northern Great Plains is an increasing threat to GR cropping systems and conservation tillage practices common in this region. GR kochia accessions with 4.6- to 11-fold levels of resistance to glyphosate have recently been reported in Montana. Those GR kochia accessions were also suspected to be resistant to acetolactate synthase (ALS) inhibitors, i.e., multiple herbicide-resistant (MHR) kochia. In this research, the level of resistance to the ALS-inhibitor herbicides (sulfonylureas) and the molecular mechanisms conferring resistance to glyphosate and ALS-inhibitor herbicides in MHR kochia was investigated. On the basis of whole-plant dose–response assays, MHR kochia accessions (GIL01, JOP01, and CHES01) were 9.3- to 30-fold more resistant to premixed thifensulfuron methyl tribenuron methyl metsulfuron methyl than the susceptible (SUS) accession. In an in vivo leaf-disk shikimate assay, MHR plants accumulated less shikimate than the SUS plants at a discriminate dose of 100 μM glyphosate. Sequencing of the conserved region of EPSPS revealed no target-site mutation at Thr₁₀₂ or Pro₁₀₆ residue. MHR kochia accessions had increased relative EPSPS gene copies (~ 4 to 10) compared with the SUS accession (single copy). Furthermore, MHR kochia accumulated higher EPSPS protein compared with the SUS plants. Resistance to the ALS-inhibitor herbicides was conferred by Pro₁₉₇ amino acid substitution (proline to glutamine). EPSPS gene amplification and a single target-site mutation at Pro₁₉₇ in ALS gene confer resistance to glyphosate and ALS-inhibitor herbicides, respectively, in MHR kochia accessions from Montana. This is the first confirmation of occurrence of MHR kochia in Montana.

Nomenclature: Glyphosate; metsulfuron methyl; thifensulfuron methyl; tribenuron methyl; Kochia, Kochia scoparia (L.) Schrad.