Ammannia multiflora Roxb. is a dominant broadleaf weed that is a serious problem in southern China rice fields, and acetolactate synthase (ALS)-inhibiting herbicides have been used for its control for more than 20 years. Excessive reliance on ALS-inhibiting herbicides has led to herbicide resistance in A. multiflora. In this study, 10 A. multiflora populations from the Jiangsu Province of China were collected, and the resistance levels and target site–resistance mechanisms to ALS-inhibiting herbicides bensulfuron-methyl and penoxsulam were investigated. The dose–response assays showed that eight populations evolved resistance to bensulfuron-methyl (9.1- to 90.9-fold) and penoxsulam (5.0- to 103.1-fold). Amplification of ALS genes indicated that there were three ALS genes (AmALS1, AmALS2, and AmALS3) in A. multiflora. Sequence analysis revealed amino acid mutations at Pro-197 in either AmALS1 (Pro-197-Ala, Pro-197-Ser, and Pro-197-His) or AmALS2 (Pro-197-Ser and Pro-197-Arg) in resistant populations, and no mutations were found in AmALS3. Moreover, two independent mutations (Pro-197-Ala in AmALS1 and Pro-197-Ser in AmALS2 or Pro-197-Ala in AmALS1 and Pro-197-Arg in AmALS2) coexisted in two resistant populations, respectively. In addition, the auxin mimic herbicides MCPA and florpyrauxifen-benzyl, the photosystem II inhibitor bentazon, and the protoporphyrinogen oxidase inhibitor carfentrazone-ethyl can effectively control the resistant A. multiflora populations. Our study demonstrates the wide prevalence of ALS inhibitor–resistant A. multiflora populations in Jiangsu Province and the diversity of Pro-197 mutations in ALS genes and provides alternative herbicide options for controlling resistant A. multiflora populations.