Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is a dominant weed species occurring in rice fields across China. Metamifop, a common herbicide, is frequently applied to control E. crus-galli and other grassy weeds in rice fields. Herein, HS01, an E. crus-galli population suspected to be resistant (R) to metamifop, was collected from Hanshan County in Anhui Province. Whole-plant dose-response testing revealed that, compared with the susceptible (S) population FD03, HS01 had developed high-level resistance to metamifop with a resistance index (RI) of 11.76 and showed cross-resistance to cyhalofop-butyl (RI=9.33), fenoxaprop-P-ethyl (RI=5.80) and clethodim (RI=3.24). Gene sequencing revealed a Cys-2088-Arg mutation in ACCase 1,5 allele of all the R plants, while ACCase gene overexpression was not involved in the resistance. Molecular docking indicated that the less negative binding energies might be the main reason for the resistance of HS01 to ACCase-inhibiting herbicides. A derived cleaved amplified polymorphic sequence (dCAPS) method was developed for the rapid identification of the Cys-to-Arg mutation in the ACCase gene at codon position 2088 in E. crus-galli. Additionally, pretreatment with the cytochrome P450 inhibitor piperonyl butoxide or the glutathione S-transferase inhibitor 4-chloro-7-nitrobenzoxa-diazole had no significant effects (p> 0.05) on the resistance of HS01 to metamifop. To our knowledge, this is the first report of a Cys-2088-Arg mutation in E. crus-galli ACCase that confers cross-resistance to ACCase-inhibiting herbicides.