The rapid evolution and spread of glyphosate-resistant (GR) kochia in theNorthern Great Plains is an increasing threat to GR cropping systems andconservation tillage practices common in this region. GR kochia accessionswith 4.6- to 11-fold levels of resistance to glyphosate have recently beenreported in Montana. Those GR kochia accessions were also suspected to beresistant to acetolactate synthase (ALS) inhibitors, i.e., multipleherbicide-resistant (MHR) kochia. In this research, the level of resistanceto the ALS-inhibitor herbicides (sulfonylureas) and the molecular mechanismsconferring resistance to glyphosate and ALS-inhibitor herbicides in MHRkochia was investigated. On the basis of whole-plant dose–response assays,MHR kochia accessions (GIL01, JOP01, and CHES01) were 9.3- to 30-fold moreresistant to premixed thifensulfuron methyl + tribenuron methyl +metsulfuron methyl than the susceptible (SUS) accession. In an in vivoleaf-disk shikimate assay, MHR plants accumulated less shikimate than theSUS plants at a discriminate dose of 100 μM glyphosate. Sequencing of theconserved region of EPSPS revealed no target-site mutationat Thr102 or Pro106 residue. MHR kochia accessions hadincreased relative EPSPS gene copies (~ 4 to 10) comparedwith the SUS accession (single copy). Furthermore, MHR kochia accumulatedhigher EPSPS protein compared with the SUS plants. Resistance to theALS-inhibitor herbicides was conferred by Pro197 amino acidsubstitution (proline to glutamine). EPSPS geneamplification and a single target-site mutation at Pro197 in ALS gene confer resistance to glyphosate andALS-inhibitor herbicides, respectively, in MHR kochia accessions fromMontana. This is the first confirmation of occurrence of MHR kochia inMontana.