Multiple herbicide resistant Echinochloa phyllopogon exhibits resistance to the auxin herbicide quinclorac. Previous research observed enhanced activity of the cyanide-detoxifying enzyme β-cyanoalanine synthase (β-CAS) and reduced ethylene production in the resistant line, suggesting β-CAS-mediated cyanide detoxification and insensitivity to quinclorac stimulation as the resistance mechanisms. To investigate the molecular mechanisms of quinclorac resistance, we characterized the β-CAS genes alongside plant transformation studies. The association of β-CAS activity and ethylene production to quinclorac resistance was assayed in the F6 progeny of susceptible and resistant lines of E. phyllopogon.A single nucleotide polymorphism in a β-CAS1 intron deleted aberrantly spliced mRNAs and enhanced β-CAS activity in the resistant line. The enhanced activity, however, was not associated with quinclorac resistance in F6 lines. The results were supported by lack of quinclorac resistance in Arabidopsis thaliana expressing E. phyllopogon β-CAS1 and no difference in quinclorac sensitivity between β-CAS knockout and wild type rice. Reduced ethylene production co-segregated with quinclorac resistance in F6 lines which were previously characterized to be resistant to other herbicides by enhanced metabolism.β-CAS does not participate in quinclorac sensitivity in E. phyllopogon. Our results suggest that mechanism(s) leading to reduced ethylene production is behind the resistance. This article is protected by copyright. All rights reserved.