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Monochoria vaginalis (Burm. f.) C. Presl, belonging to the family Pontederiaceae, is an aquatic herbaceous plant, native to temperate and tropical Asia. The species often occurs in paddy fields as a noxious weed in East Asia, and in the USA, and causes a significant reduction in rice production. The objective of the present research was the evaluation of the resistance levels of M. vaginalis against three chemical groups of acetolactate synthase (ALS)-inhibitor herbicides and other two different groups of herbicides, and the investigation of the mutations in the ALS gene of the resistant biotype of M. vaginalis. Herbicide dose–response experiments showed that the resistant biotype of M. vaginalis was highly resistant to bensulfuron-methyl, moderately resistant to bispyribac-sodium, had low resistance to penoxsulam and 2,4-D dimethyl ammonium, and was susceptible to sulfentrazone. The nucleotide sequences of the ALS gene of resistant and susceptible biotypes showed 14 base substitutions, which caused two amino acid substitutions: Val-143-Ile and Val-148-Ile. It is the first report of the substitutions of amino acids Val-143-Ile and Val-148-Ile in ALS protein. Those mutations may give different resistance spectra against three ALS-inhibitor herbicides: bensulfuron-methyl, bispyribac-sodium, and penoxsulam. Further research is needed to elucidate the molecular basis of target-site resistance mechanisms such as the transformation of the ALS gene of M. vaginalis. It is also necessary to evaluate herbicide mixtures and/or the rotation of herbicide sites of action to control the resistant biotype of M. vaginalis. [ABSTRACT FROM AUTHOR]