Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is a dominant weed species occurring in rice (Oryza sativa L.) 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, China. 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 the 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 acetyl-CoA carboxylase (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-nitrobenzoxadiazole 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.