Echinochloa crus-galli is a major weed in rice fields in China, and quinclorac has been long used for its control. Over-reliance of quinclorac has resulted in quinclorac resistance in E. crus-galli. Two resistant (R) E. crus-galli populations from Hunan, China were confirmed to be at least 78-fold more resistant to quinclorac than the susceptible (S) population. No difference in foliar uptake of 14C-labelled quinclorac was detected between the R and S plants. However, a higher level of 14C translocation and a lower level of quinclorac metabolism were found in the R plants. Basal and induced expression levels of β-cyanoalanine synthase (β-CAS) gene and β-CAS activity were not significantly different between the R and S plants. However, the induction expression of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1) gene by quinclorac treatment was evident in the S plants but not in the R plants. Quinclorac resistance in the two resistant E. crus-galli populations was not likely to be related to foliar uptake, translocation or metabolism of quinclorac, nor to cyanide detoxification via β-CAS. Thus, target-site based quinclorac signal reception and transduction and regulation of the ethylene synthesis pathway should be the focus for further research.