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Monochoria korsakowii is an increasingly significant threat to rice production across China, particularly in Liaoning province. Few studies have reported herbicide resistance in M. korsakowii , and resistance status and mechanisms are poorly understood. Here, thirty field populations of M. korsakowii were collected from 11 rice-growing regions of Liaoning, and 97% of populations had evolved resistance to bensulfuron-methyl (BM), with majority (24 of 28) showing high resistance levels (RI > 10). The first in-depth analysis of molecular features of AHAS1 and AHAS2 in BM-resistant populations showed that four Pro197 mutations (Pro197 to His, Ala, Leu or Ser) in AHAS1 and one mutation (Pro197Ser) in AHAS2 were identified. Notably, novel double Pro197Ser mutations co-occurred in both AHAS1 and AHAS2 in the most resistant line LN-20. Furthermore, resistant mutants were used to investigate the effect of Pro197 mutations on AHAS functionality, binding modes, gene expression and cross-resistance in M. korsakowii. All the detected Pro197 mutations considerably reduced in vitro AHAS sensitivity to BM by weakening hydrogen bonds and hydrophobic interactions in the predicted BM-AHAS complexes, especially the double Pro197Ser mutations. This novel resistance mutation combination slightly impacted the extractable AHAS activity, and increased the affinity and catalytic rate of pyruvate. Also, the AHAS expression level was significantly up-regulated. Moreover, all mutations provided resistance only to other sulfonylureas herbicides but not triazolopyrimidine or pyrimidinyl-benzoates herbicides. In conclusion, bensulfuron-methyl resistance in M. korsakowii was grim in Liaoning, China, and amino acid mutations on AHAS isozymes were the primary resistance mechanism. Double Pro197Ser mutations in both AHAS1 and AHAS2 confer higher herbicide resistance than single mutations in AHAS1. Thus, this work deepens our understanding of resistance status and mechanisms of M. korsakowii. [Display omitted] • Target-site mutations on AHAS isozymes were the primary resistance mechanism of M. korsakowii in Liaoning, China. • Four Pro197 mutations (Pro197 to His, Ala, Leu or Ser) in AHAS1 and one mutation (Pro197Ser) in AHAS2 were identified in M. korsakowii. • Pro197 mutations show different functional impacts on AHAS functionality, binding modes, gene expression and cross resistance. • Double Pro197Ser mutations in both AHAS1 and AHAS2 confer higher herbicide resistance than single mutations in AHAS1. [ABSTRACT FROM AUTHOR]