Shortawn foxtail (Alopecurus aequalis Sobol.) is an obligate wetland plant that is widely distributed throughout Europe, temperate Asia, and North America. In China, it is widespread in the middle and lower reaches of the Yangtze River as a noxious weed in winter cropping fields with a rice (Oryza sativa L.) rotation. The acetolactate synthase (ALS)-inhibiting herbicide mesosulfuron-methyl has been widely used to control annual grass and broadleaf weeds, including A. aequalis, in wheat (Triticum aestivum L.) fields, leading to the selection of herbicide-resistant weeds. In this study, an A. aequalis population, AHFT-4, that survived mesosulfuron-methyl at the field-recommended rate (9 g ai ha^–1) was collected in Anhui Province. Single-dose testing confirmed that the suspected resistant AHFT-4 had evolved resistance to mesosulfuron-methyl. Target gene sequencing revealed a resistance mutation of Pro-197-Ala in ALS1 of the resistant plants, and a derived cleaved amplified polymorphic sequence marker was developed to specifically detect the mutation. A relative expression assay showed no significant difference in ALS expression between AHFT-4 and a susceptible population without or with mesosulfuron-methyl treatment. Whole-plant dose–response bioassays indicated that AHFT-4 had evolved broad-spectrum cross-resistance to ALS-inhibiting herbicides of all five chemical families tested, with GR₅₀ resistance index (RI) values ranging from 21 to 206. However, it remained susceptible to the photosystem II inhibitor isoproturon. Pretreatment with the cytochrome P450 inhibitor malathion or the glutathione S-transferase inhibitor 4-chloro-7-nitrobenzoxa-diazole had no significant effects on the resistance of AHFT-4 to mesosulfuron-methyl. To our knowledge, this study reports for the first time the ALS gene Pro-197-Ala substitution conferring broad-spectrum cross-resistance to ALS-inhibiting herbicides in A. aequalis.