Keng stiffgrass is a grass weed that affects wheat-rice cropping systems in China.The extensive reliance on acetyl coenzyme Acarboxylase(ACCase)-inhibiting herbicides has resulted in keng stiffgrass developing resistance to these herbicides.The objective of this research was to evaluate the resistance level of the putative resistant keng stiffgrass populations to ACCase-inhibiting herbicides and to identify their molecular resistance mechanism.Whole-plant dose-response experiments demonstrated that SD-4(R),SD-11(R),and JS-25(R) populations were highly resistant to fenoxaprop,clodinafop,and fluazifop,moderately resistant to diclofop,had low resistance to sethoxydim and pinoxaden,but were sensitive to clethodim.Partial chloroplastic ACCase sequences showed that there were two copies of ACCase gene in keng stiffgrass,and all homoeologous genes were expressed.The results of sequence analyses of the ACCase CT domain revealed an isoleucine-to-asparagine substitution at position 2041 in SD-4(R) and SD-11(R) populations,and a tryptophan-to-cysteine substitution at position 2027 in the JS-25(R) population.To our knowledge,this is the first report of lle-2041-Asn and Trp-2027-Cys mutations in ACCase-resistant keng stiffgrass.In addition,three robust(derived) cleaved amplified polymorphic sequence((d)CAPS) markers have been developed to rapidly identify these mutations in the ACCase gene of keng stiffgrass. Keng stiffgrass is a grass weed that affects wheat-rice cropping systems in China. The extensive reliance on acetyl coenzyme Acarboxylase (ACCase) -inhibiting herbicides has resulted in keng stiffgrass developing resistance to these herbicides.The objective of this research was to evaluate the resistance level of the putative resistant keng stiffgrass populations to ACCase-inhibiting herbicides and to identify their molecular resistance mechanism. Well-plant dose-response procedures demonstrated that SD-4 (R), SD-11 (R), and JS- 25 ) populations were highly resistant to fenoxaprop, clodinafop, and fluazifop, moderately resistant to diclofop, had low resistance to sethoxydim and pinoxaden, but were sensitive to clethodim. Partially chloroplastic ACCase sequences showed that there were two copies of ACCase gene in keng stiffgrass, and all homoeologous genes were expressed. The results of sequence analyzes of the ACCase CT domain revealed an isoleucine-to-asparagine substitution at position 2041 in SD-4 ( R) and SD-11 (R) populations, and a tryptophan-to-cysteine ​​substitution at position 2027 in the JS-25 population.Tho our knowledge, this is the first report of lle-2041- Asn and Trp- 2027-Cys mutations in ACCase-resistant keng stiffgrass.In addition, three robust (derived) cleaved amplified polymorphic sequences ((d) CAPS) markers have been developed to identify these mutations in the ACCase gene of keng stiffgrass.