American sloughgrass [Beckmannia syzigachne (Steud.) Fernald] is one of the most predominant and troublesome weeds in wheat (Triticum aestivum L.) fields rotated with rice (Oryza sativa L.) in China. Mesosulfuron-methyl is one of the main herbicides used to selectively control B. syzigachne in winter wheat fields in China. After many years of application, mesosulfuron-methyl failed to control B. syzigachne in Yutai County. The objectives of this study were to determine the resistance level to mesosulfuron-methyl and other acetolactate synthase (ALS) inhibitors in the B. syzigachne population collected from Yutai County (R) and identify the mechanism of resistance. The results indicated that the R population was 4.1-fold resistant to mesosulfuron-methyl and was cross-resistant to pyroxsulam (600-fold), imazethapyr (4.1-fold), flucarbazone (12-fold), and bispyribac-sodium (12-fold). In vitro assays revealed that ALS in the R population was as sensitive as that in a susceptible (S) population. Gene sequence analysis identified no known resistant mutations in the ALS gene of the R population. Furthermore, real-time quantitative reverse transcriptase PCR experiments indicated that the expression level of the ALS gene in the R population was not different from that of the S population. However, the cytochrome P450 inhibitor malathion reversed the R population's resistance to mesosulfuron-methyl. The result of ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS-MS) spectral analysis indicated that the metabolic rates of mesosulfuron-methyl in the R population were significantly faster than in the S population. Therefore, non-target resistance to mesosulfuron-methyl has been demonstrated in the R population. The resistance was very likely caused by enhanced herbicide metabolism.