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Chinese sprangletop [Leptochloa chinensis (L.) Nees] is one grass weed severely affecting rice growth in paddies in China. Cyhalofop-butyl is the main herbicide used to control grass weeds in Chinese paddy fields, especially for controlling L. chinensis; however, L. chinensis has evolved resistance to cyhalofop-butyl due to continuous and extensive application. To investigate cyhalofop-butyl resistance levels and mechanisms in L. chinensis in some of the Chinese rice areas, 66 field populations were collected and treated with cyhalofop-butyl. Of these tested populations, 10 showed a high level of resistance to cyhalofop-butyl; the 50% effective dose (GR50) ranged within 108.4 to 1443.5 g ai ha–1 with resistance index (RI) values of 9.1 to 121.8 when compared to the susceptible population. Acetyl-coenzyme A carboxylase (ACCase) genes of susceptible and all 10 resistant populations were amplified and sequenced. Among them, Ile-1781-Leu, Trp-2027-Cys, Trp-2027-Ser, and Ile-2041-Asn mutations were found in five resistant populations. No known resistance-related mutations were found in the other five resistant populations, indicating that resistance to cyhalofop-butyl in these populations was likely to be endowed by non-target-site resistance mechanisms. Notably, the Ile-1781-Leu and Trp-2027-Cys substitutions have previously been reported, but this is the first report of Trp-2027-Ser and Ile-2041-Asn mutations in L. chinensis. Furthermore, three derived cleaved amplified polymorphic sequence (dCAPS) methods were developed to rapidly detect these mutations in L. chinensis.