Chinese sprangletop (Leptochloa chinensis (L.) Nees) is a serious grass weed in rice paddies. In some areas, L. chinensis has become resistant to the herbicide cyhalofop-butyl because of its frequent and extensive use over the past five years. In this study, whole-plant dose-response assays were conducted, and a L. chinensis population (ZHYH) had a 75.8-fold resistance index to cyhalofop-butyl. Molecular analyses revealed that this resistance was attributed to a tryptophan (Trp)-2027-to-cysteine (Cys) substitution in the CT domain of the ACCase gene. To our knowledge, this is the first report revealing the mechanism underlying cyhalofop-butyl resistance in L. chinensis. Furthermore, a derived cleaved amplified polymorphic (dCAPS) assay was developed to rapidly detect the Trp-2027-Cys mutation. Of the 100 ZHYH plants analyzed, 52 were heterozygous mutants and 48 were susceptible homozygous plants. In addition, the cyhalofop-butyl-resistant L. chinensis was cross-resistant to aryloxyphenoxypropionate and phenylpyrazoline herbicides, but not to cyclohexanedione, acetolactate synthase-inhibiting, protoporphyrinogen oxidase, and urea herbicides, and had only slight resistance to the hormonal herbicide quinclorac.