Abstract: Shortawn foxtail (Alopecurus aequalis Sobol.) is one of the major malignant weeds infesting wheat (Triticum aestivum L.) fields with a rice (Oryza sativa L.) rotation across the middle and lower reaches of the Yangtze River in China. Mesosulfuron-methyl as an important herbicide is widely used for control of A. aequalis and other grass weeds in wheat fields. Previously, a suspected herbicide-resistant A. aequalis population AHFT-01 was collected from a wheat field in Fengtai County, Anhui Province, China. To clarify its herbicide resistance status and potential resistance mechanisms, greenhouse pot experiments at the whole-plant level were performed to determine the susceptibility levels of AHFT-01 to mesosulfuron-methyl and several other acetolactate synthase (ALS) inhibitors. And different copies of the full-length ALS genes of the resistant and susceptible populations of A. aequalis were amplified, sequenced, and aligned. The results showed that compared with the susceptible population AHHN-02, A. aequalis population AHFT-01 was highly resistant to mesosulfuron-methyl with a resistance index (RI) of 28.50. ALS gene sequencing revealed that 100% of the plants of AHFT-01 carried the homozygous Pro (CCC)-197-Thr (ACC) mutation in their ALS1 alleles. AHFT-01 plants also conferred different levels of cross-resistance to imazethapyr, pyroxsulam, and flucarbazone-sodium, with RI values of 8.18, 10.15, and 10.13, respectively. In addition, AHFT-01 was lowly resistant to the acetyl-CoA carboxylase (ACCase) inhibitor pinoxaden but was susceptible to the other two ACCase inhibitors including fenoxaprop-P-ethyl and clethodim and the photosynthetic II (PSII) inhibitor isoproturon. This work indicates that the amino acid substitution at position 197 of ALS1 allele may be one of the important reasons that caused targe-site based resistance to mesosulfuron-methyl in A. aequalis.