Bearded sprangletop is a problematic native grass weed in California’s rice fields. The widespread and extensive use of Acetyl-CoA carboxylase (ACCase) inhibiting herbicides, such as cyhalofop-P-butyl (cyhalofop), has led to speculation that biotypes of bearded sprangletop have developed herbicide resistance to ACCase. The aim of this study was to evaluate suspected resistant bearded sprangletop biotypes, R1, R2, R3, and the susceptible biotype, S1, in terms of their levels of resistance to three ACCase-inhibiting herbicides and to characterize the molecular mechanisms of resistance. Dose-response experiments suggested that the biotype R1, R2, and R3 had high-level resistance to cyhalofop, and quizalofop-P-ethyl (quizalofop), but not clethodim. It was identified that the resistance to ACCase inhibitors was a target site mechanism resulting from nucleotide substitution. The carboxyl transferase (CT) domain of the ACCase gene’s sequence analysis revealed the substitutions Trp-2027-Cys for R1 and R2 biotypes and Ile-2041-Asn for R3 biotype. This study revealed that presence of target-site resistance to cyhalofop and quizalofop in at least two mutation points in representative biotypes of bearded sprangletop in California. This research highlights the significance of careful herbicide selection due to weed species responding quite rapidly to selection pressure to help manage bearded sprangletop in rice field.