American sloughgrass is a troublesome grass weed in winter wheat fields after rice in China. Mesosulfuron-methyl failed to control American sloughgrass in Danyang County in 2012. The purpose of this research was to determine the resistance level to mesosulfuron and other herbicides in American sloughgrass and to identify the molecular basis of resistance. Dose–response experiments indicated that this population was moderately resistant to mesosulfuron-methyl (7.6-fold) and pyroxsulam (6.0-fold), highly resistant to flucarbazone-sodium (20.3-fold), fenoxaprop-p-ethyl (565.0-fold), clodinafop-proargyl (19.5-fold), and pinoxaden (45.9-fold), and susceptible to isoproturon. Part of the acetolactate sythase (ALS) gene was cloned and sequenced to confirm the molecular mechanism of resistance to ALS-inhibiting herbicides. A Pro197Ser substitution was identified. This substitution is likely the molecular mechanism of resistance to mesosulfuron-methyl in the Danyang population in which it is cross-resistant to flucarbazone-sodium and pyroxsulam. This study established the first report of mesosulfuron-methyl resistance likely caused by a Pro197 substitution in American sloughgrass and a potential herbicide to control this resistant weed.