Abstract: In order to investigate the resistance level and molecular mechanism of Ammannia multiflora Roxb. in paddy fields to acetolactate synthase (ALS) inhibiting herbicide bensulfuron-methyl, the resistance index (RI) of a putative resistant population YZ-R collected from Yangzhou city were performed by the whole-plant dose-response experiments. In vitro ALS enzyme sensitivity to bensulfuron-methyl and the nucleotide sequence of ALS gene from the susceptible population YZ-S and resistant population YZ-R were analyzed to characterize the target-site resistance mechanism. The whole-plant dose-response assays indicated that the YZ-R population had evolved a high level (RI = 40.6) of resistance to bensulfuron-methyl. In addition, the I₅₀ values of YZ-R and YZ-S were 0.087 and 0.0028 μmol/L, respectively, and the RI was 31.1. The partial sequence of the target ALS gene was cloned by PCR, which contains eight reported amino acid mutation sites. Sequence alignment results showed the substitution of proline (Pro) 197 by serine (Ser) in the YZ-R population. This study demonstrated that the reduced ALS enzyme sensitivity to bensulfuron-methyl resulted from the Pro-197-Ser substitution in the ALS gene, which was the main cause of the high-level resistance to bensulfuron-methyl in A. multiflora.